JP2008535836A - Methods and compositions for the treatment of anxiety disorders - Google Patents

Abstract

The present invention is ^Na + ^-K + -2Cl ^- reducing the effective amount of (NKCC) symporter, inactivating, and / or by administering the agent that is effective in inhibiting the activity , Methods and compositions for treating neuropathic pain and neuropsychiatric disorders. In certain ^embodiments, Na ⁺ -K ⁺ -2Cl ^- cotransporter is NKCC1. The compositions and methods of the present invention do not suppress neuronal excitability, thus avoiding the undesirable side effects often associated with drugs currently used for the treatment of neuropathic pain and neuropsychiatric disorders. It can be used to reduce neuronal hypersynchronization and / or neuropsychiatric disorders associated with disabling pain.

Description

REFERENCES TO RELATED APPLICATIONS This application is filed in US patent application Ser. No. 11 / 251,724 (filed Oct. 17, 2005); U.S. Patent Application No. 11 / 130,945 (filed May 17, 2005); And claims priority to US patent application Ser. No. 11 / 101,000 (filed Apr. 7, 2005).

The present invention relates to methods and compositions for treating selected conditions of the central and peripheral nervous system using non-synaptic mechanisms. More particularly, the invention relates to methods and compositions for treating neuropsychiatric disorders by administering agents that modulate the expression and / or activity of the sodium-potassium-chloride cotransporter.

BACKGROUND OF THE INVENTION Neuropathic pain and nociceptive pain differ in their etiology, pathophysiology, diagnosis and treatment. Nociceptive pain occurs in response to activation of a specific subset of peripheral sensory neurons, namely nociceptors. It is generally acute (except for joint pain) and self-limiting and serves a protective biological function by acting as a warning of ongoing tissue damage. It is typically clearly localized and often has the nature of aching or pulsating pain. Examples of nociceptive pain include postoperative pain, sprains, fractures, burns, bruises, inflammation (from infection or joint damage), obstruction and fascial pain. Nociceptive pain can usually be treated with opioids and non-steroidal anti-inflammatory drugs (NSAIDs).

  Neuropathic pain is a common type of chronic non-malignant pain, which is the result of injury or dysfunction in the peripheral or central nervous system and does not serve a protective biological function. It is estimated that over 1.6 million people are affected in the US population. Neuropathic pain has many different etiologies and includes, for example, trauma, diabetes, herpes zoster infection, HIV / AIDS peripheral neuropathy, terminal cancer, amputation (including mastectomy), carpal tunnel It can be caused by syndromes, chronic alcohol intake, exposure to radiation and unintended side effects of neurotoxic therapies such as certain anti-HIV and chemotherapeutic agents.

  In contrast to nociceptive pain, neuropathic pain is often described as “burning”, “electrical”, “prickling”, or “stab”. It is characterized by chronic allodynia (defined as pain resulting from stimuli that do not normally trigger a pain response, such as light contact) and hyperalgesia (usually defined as increased sensitivity to painful stimuli). Often, and any damaged tissue may persist for months or years after apparent healing.

  Neuropathic pain is difficult to treat. Analgesics that are effective for normal pain (eg, opioid narcotics and non-steroidal anti-inflammatory drugs) are rarely effective for neuropathic pain. Similarly, agents that have activity in neuropathic pain are usually not effective for nociceptive pain. Standard drugs used to treat neuropathic pain often act selectively in alleviating certain symptoms but not others in some patients (eg allodynia is alleviated) But hyperalgesia is not relieved). For this reason, a combination of different drugs and individually adapted therapies may be required for successful treatment (eg Bennett, Hosp. Pract. (Off Ed). 33: 95-98). , 1998). Therapeutic agents typically used in the management of neuropathic pain are tricyclic antidepressants (eg amitriptyline, imipramine, desimipramine and clomipramine), generalized local anesthetics and anticonvulsants (eg phenytoin, carbamazepine, valproic acid, clonazepam) And gabapentin).

  Many anticonvulsants originally developed for the treatment of epilepsy and other seizure disorders have applications in the treatment of non-maniac conditions such as neuropathic pain, mood disorders (eg bipolar affective disorders) and schizophrenia (See Rogawski and Loscher, Nat. Medicine, 10: 685-692, 2004 for a discussion of the use of antiepileptics in the treatment of non-manic states). That is, epilepsy, neuropathic pain and affective disorder are common pathophysiological mechanisms (Rogawaski & Loscher, supra; Ruschweyh & Sandkuhler, Pain 105: 327-338, 2003), ie pathological increase in neuronal excitability And a corresponding inappropriately high frequency of spontaneous neuron firing. However, only some but not all anti-epileptics are effective in treating neuropathic pain, and furthermore, such anti-epileptics are effective in certain subsets of patients with neuropathic pain (McCleane, Expert. Opin. Pharmacother. 5: 1299-1312, 2004).

  Spiders are characterized by an abnormal discharge of cerebral neurons and are typically manifested as various types of seizures. Spider-like activity is discovered by spontaneously generated synchronized discharges of neuronal populations that can be measured using electrophysiological techniques. This synchronized activity that discriminates between moth-like activity and non-cocoon-like activity is called “over-synchronization” because it can cause individual neurons to discharge in a time-locked manner relative to each other. This is to explain the increasing state. Hypersynchronous activity is typically induced in the experimental model of epilepsy by increasing excitatory synaptic currents or decreasing inhibitory, and thus hyperexcitation itself is responsible for the generation and maintenance of epileptiform activity. It was presumed to be the decisive feature involved. Similarly, neuropathic pain was thought to involve the conversion of neurons involved in the transmission of pain from the normal susceptibility state to the hypersensitive one (Costigan & Woolf, Jnl. Pain 1: 35-). 44, 2000). Thus, the focus in developing treatments for both epilepsy and neuropathic pain is (a) suppresses action potential generation; (b) increases inhibitory synaptic transmission; or (c) excitability. It was to suppress neuronal hyperexcitability by either reducing synaptic transmission. However, hypersynchronous epileptiform activity is dissociated from hyperexcitability, and the cation chloride cotransport inhibitor furosemide has been shown to reversibly block synchronous discharge without reducing the hyperexcited synaptic response ( Hochman et al. Science 270: 99-102, 1995).

  Abnormal expression of the sodium channel gene (Waxman, Pain 6: S133-140, 1999; Waxman et al. Proc. Natl. Acad. Sci USA 96: 7635-7639, 1999) and pacemaker channel (Chaplan et al. J. Neurosci). 23: 1169-1178, 2003) both play a molecular role in neuropathic pain.

  Neuropsychiatric disorders, such as anxiety disorders, are generally treated with counseling and / or medication. Most of the drugs currently used in the treatment of such disorders have significant negative side effects, such as dependence-inducing tendency.

The cationic chloride cotransporter (CCC) is an important regulator of neuronal chloride concentration that is thought to affect various aspects of cell-to-cell communication and neuronal development, formation and trauma. The CCC gene family consists of three large groups: Na ⁺ -Cl ^- cotransporter (NCC), K ⁺ -Cl ^- cotransporter (KCC) and Na ⁺ -K ⁺ -2Cl ^- cotransporter (NKCC). . Two NKCC isoforms have been discovered, NKCC1 is present in a wide variety of secretory and non-epithelial cells, and NKCC2 is primarily expressed in the kidney. For a discussion on the structure, function and regulation of NKCC1, see Haas and Forbush, Annu. Rev. Physiol. 62: 515-534, 2000. Randall et al. Have discovered two splice variants of the Slc12a2 gene encoding NKCC1 designated NKCC1a and NKCC1b (Am. J. Physiol. 273 (Cell Physiol. 42): C1267-1277, 1997). The NKCC1a gene has 27 exons, while the splice variant NKCC1b lacks exon 21. NKCC1b splice variants are mainly expressed in the brain. NKCC1b is thought to be more than 10% more active than BKCC1a, but it is proportionally present in the brain in much smaller amounts than NKCC1a. It has been suggested that differential splicing of the NKCC1 transcript plays a regulatory role in human tissues (Vibat et al. Anal. Biochem. 298: 218-230, 2001). Na-K-Cl cotransport in all cells and tissues is suppressed by loop diuretics such as furosemide, bumetanide and benzmethanide.

Na-K-2Cl cotransporter knockout mice have been shown to have an impaired nociceptive phenotype and abnormal gait and motility (Non-Patent Document 1). Delpire and Mount suggest that NKCC1 is involved in pain perception (Non-Patent Document 2). Laird et al. Recently reported a study showing reduced hyperalgesia during caressing in NKCC1 knockout mice when compared to wild type and heterozygous mice (3). However, in this acute pain model, no difference in discontinuous hyperalgesia has been observed between the three groups of mice. Morales-Aza et al. Show that altered expression of NKCC1 and K-Cl cotransporter KCC2 contributes to the control of spinal excitability in arthritis and is thus a therapeutic target for the treatment of inflammatory pain (Non-Patent Document 4). Granados-Soto et al. Reported a rat test in which formalin-induced nociception was reduced by administration of the NKCC inhibitor bumetanide, furosemide or piretanide (Non-patent Document 5). The formalin-induced acute pain model is widely used, but is considered to have little relevance to the chronic pain state (Non-Patent Document 6). Simultaneous treatment of brain damage induced by episodic alcohol exposure using NMDA receptor antagonists, non-NMDA receptors and Ca ²⁺ channel antagonists with furosemide prevents alcohol hydration and electrolyte rise while preventing alcohol dependence It is known to reduce cerebral cortical damage by 75 to 85% (Non-patent Document 7). According to the authors, the results suggest that furosemide and related drugs may be useful as neuroprotective agents in alcohol abuse. Willis et al. Have reported studies showing that nedocromil sodium, furosemide and bumetanide reduce histamine-induced pruritus and redness responses in human skin in vivo by inhibiting sensory nerve activation. Espinosa et al. And Ahmad et al. Have previously suggested that furosemide is useful in the treatment of certain types of epilepsy (Non-Patent Document 8; and Non-Patent Document 9).

As with epilepsy, the focus of pharmacological intervention in neuropathic pain was to reduce neuronal hyperexcitation. The vast majority of drugs currently used to treat neuropathic pain, for example, modulate the release or activity of excitatory neurotransmitters, enhance inhibitory pathways, ion channels involved in impulse generation It targets synaptic activity in the excitatory pathway by blocking and / or functioning as a membrane stabilizer. That is, conventional drugs and treatment methods for the treatment of neuropathic pain and neuropsychiatric disorders reduce neuronal excitability and suppress synaptic firing. One significant difficulty with these therapeutic agents is that they are non-selective and show their effects on both normal and abnormal neuronal populations. This results in unintended negative side effects, which can affect normal CNS functions such as cognition, learning and memory and have adverse physiological and psychological effects in the patient being treated. Bring. Common side effects include excessive calming, drowsiness, loss of memory and liver damage. Accordingly, there remains a need for methods and compositions for the treatment of neuronal disorders that disrupt hypersynchronous neuronal activity without compromising neuronal excitability and spontaneous synchronization required for normal functioning of the peripheral and central nervous systems. Has been.
Sung et al. Jnl. Neurosci. 20: 7531-7538,2000 Ann. Rev. Physiol. 64: 803-843, 2002 Neurosci. Letts. 361: 200-203, 2004 Neurobiol. Dis. 17: 62-69, 2004 Pain 114: 231-238, 2005 Walker et al. Mol. Med. Today 5: 319-321, 1999 Collins et al, FASEB J. et al. 12: 221-230, 1998. Medicina Espanola 61: 280-281, 1969 Brit. J. et al. Clin. Pharmacol. 3: 621-625, 1976

SUMMARY OF THE INVENTION Therapeutic compositions and methods of the present invention are characterized by conditions including neuropathic pain and neuropsychiatric disorders, such as bipolar disorder, anxiety disorders (eg, panic disorder, social anxiety disorder, obsessive disorder). Treat sexual disorders, post-traumatic stress disorder, generalized anxiety disorder and specific phobia (American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th edition, 2000 revision), depression and schizophrenia Useful for. The compositions and methods of the present invention do not suppress neuronal excitability, thus avoiding the undesirable side effects often associated with drugs currently used for the treatment of neuropathic pain and neuropsychiatric disorders. It can be used to reduce neuronal hypersynchronization and / or neuropsychiatric disorders associated with disabling pain.

The methods and compositions disclosed herein generally use non-synaptic mechanisms and modulate, generally reduce, synchronization of neuronal population activity. Synchronization of neuronal population activity is modulated by manipulating the concentration and gradient of central and / or peripheral nervous system anions. More particularly, the compositions of the present invention may reduce, inactivate, and / or inhibit activity of an effective amount of Na ⁺ -K ⁺ -2Cl ⁻ (NKCC) cotransporter. it can. Particularly preferred therapeutic agents of the present invention show a high degree of NKCC cotransporter antagonist activity in a population of central and / or peripheral nervous system cells such as glial cells, Schwann cells and / or neuronal cells, and renal cell populations Shows a lower degree of activity. In one embodiment, the composition of the invention can reduce, inactivate, and / or inhibit activity of an effective amount of the cotransporter NKCC1. NKCC1 antagonists are particularly preferred therapeutic agents for use in the methods of the present invention. NKCC cotransporter antagonists that can be commonly used in the therapeutic compositions of the invention include, but are not limited to, CNS targeted NKCC cotransporter antagonists such as furosemide, bumetanide, ethacrynic acid, tolsemide, azosemide, muzolimine, piretanide, tripamide, and the like, and Thiazide and thiazide-like diuretics such as bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylcrothiazide, polythiazide, trichloromethiazide, chlorotalidone, indapamide, metolazone and kinetazone, and analogs of such compounds And functional derivatives.

  Analogs of CNS targeting NKCC cotransporter antagonists such as furosemide, bumetanide, piretanide, azosemide and torsemide usefully used in the compositions and methods of the present invention include those described below as Formulas IV. We have found that such analogs have increased lipophilicity and reduced diuretic action compared to the CNS targeting NKCC cotransporter antagonist from which they are derived, and thus in the therapeutic methods of the present invention. Considered to reduce undesirable side effects when used.

  In one embodiment, the level of diuresis following administration of an effective amount of an analog described below as Formulas I-V is 99%, 90%, 80% of that occurring after administration of the parent molecule from which the analog is derived. %, 70%, 60%, 50%, 40%, 30%, 20% or less than 10%. For example, the analog may be less diuretic than the parent molecule when administered at the same mg / kg dose. Alternatively, an analog may be even more potent than its parent molecule so that the analog dose required for effective symptom relief is small, thereby exhibiting a low diuretic effect. Similarly, analogs may have a longer duration of action in disease treatment than the parent molecule so that the analog may be administered less frequently than the parent molecule, thereby reducing the overall diuretic effect within a given period of time. Can do.

  Other therapeutic agents usefully used in the methods and compositions of the invention include, but are not limited to, an antibody that specifically binds NKCC1, or an antigen-binding fragment thereof; a soluble NKCC1 ligand; a small molecule inhibitor of NKCC1; Antisense oligonucleotides; NKCC1-specific short interfering RNA molecules (siRNA or RNAi); and engineered soluble NKCC1 molecules. Preferably, such antibodies or antigen binding fragments thereof and small molecule inhibitors of NJCC1 are described in, for example, Haas and Forbush, Annu. Rev. Physiol. 62: 515-534, 2000 specifically binds to the domain of NKCC1 involved in bumetanide binding. The polynucleotide sequence of NKCC1 is as shown in SEQ ID NO: 1, and the corresponding cDNA sequence is shown in SEQ ID NO: 2.

  Because the methods and therapeutics of the present invention use a “non-synaptic” mechanism, little or no suppression of neuronal excitability occurs. More particularly, the therapeutics of the invention cause little (less than 1% change compared to pre-dose level) or no action potential generation or excitatory synaptic transmission suppression. In fact, a slight increase in neuronal excitability may occur upon administration of certain of the therapeutic agents of the present invention. This is in sharp contrast to the conventional antiepileptic drugs currently used to treat neuropathic pain that actually suppresses neuronal excitability. The methods and therapeutics of the present invention affect the synchronization or relative synchrony of neuronal population activity. Preferred methods and therapeutic agents modulate neuronal synchronization or relative synchrony without substantially affecting neuronal excitability by modulating the concentration and / or gradient of extracellular anionic chloride in the central or peripheral nervous system. Reduce.

  In one aspect, the present invention provides spontaneous oversynchronization of neuronal activity in specific cells and nerve fibers of the peripheral nervous system, such as primary sensory afferent fibers, pain fibers, dorsal horn neurons and sensory and pain pathways on the spinal column. It relates to methods and agents for alleviating neuropathic pain or abnormal pain perception by affecting or modulating the propagation of burst and action potentials or impulse conduction.

  The therapeutic agents of the present invention may be used in combination with other known therapeutic agents, such as those currently used for the treatment of neuropsychiatric disorders. Those skilled in the art will appreciate that combinations of therapeutic agents of the present invention with other known therapeutic agents may involve both synaptic and non-synaptic mechanisms.

  The therapeutic compositions and methods of the present invention can be used therapeutically and episodicly after the onset of symptoms or prophylactically before the onset of certain symptoms. For example, the therapeutic agents of the present invention may be used to treat existing neuropathic pain or from neurotoxic injury and neuropathic pain secondary to chemotherapy, radiation therapy, exposure to infectious agents, etc. Can be used to protect.

  In certain embodiments, the therapeutic agent used in the methods of the invention is administered using a delivery system that can cross the blood brain barrier and / or facilitate delivery of the agent to the central nervous system. For example, the permeability of the blood brain barrier to therapeutic agents can be increased transiently and reversibly by optionally using various blood brain barrier (BBB) permeability enhancers. Such BBB permeability enhancers include leukotrienes, bradykinin agonists, histamine, adhesion-breaking agents (eg zonulin), hypotonic solutions (eg mannitol), cytoskeletal contractors, short chain alkylglycerols (eg 1-O-pentyl). Glycerol) and others currently known in the art.

  These and other features of the present invention and the sun from which they are obtained are best understood by reference to the following more detailed description. All references disclosed herein are incorporated herein by reference in their entirety as though they were individually incorporated.

Detailed Description of the Invention As noted above, preferred therapeutics and methods of the present invention for use in the treatment of neuropathic pain and / or neuropsychiatric disorders are in the region of increased synchronization by reducing the activity of the NKCC cotransporter. Modulates or disrupts the synchrony of neuronal population activity. As detailed below and as described in the Examples, ion migration and ion gradient modulation using an ion-dependent symporter, preferably a cation-chloride-dependent symporter, is important for the regulation of neuronal synchronization. It is. The co-transport function of chloride has long been thought to be primarily directed to the movement of chloride out of the cell. Sodium-independent transporters known to localize to neurons move chloride ions out of neurons. Blocking this transporter, preferably by administration of the urinary furosemide, results in hyperexcitability, which is a short-term response to a cation-chloride cotransporter such as furosemide. However, the long-term response to furosemide is that the inward sodium-dependent migration of chloride ions mediated by the glial-associated Na ⁺ -K ⁺ -2Cl ^- cotransporter NKCC1 does not affect excitability and stimulus-induced cell activity It shows an active role in blocking neuron synchronization. Haglund and Hochman have shown that furosemide can block sputum activity in humans without affecting normal brain activity (J. Neurophysiol. (Feb. 23, 2005) doi: 10.1152 / jn. 00944.2004). These results support the effective use of the methods and compositions of the present invention in the treatment of neuropathic pain without the undesirable side effects often observed with conventional treatments.

  As described above, the NKCC1 splice variant called NKCC1b is more active than the NKCC1a variant. That is, the central or peripheral nervous system expressing NKCC1b at several percent higher levels is likely to be affected by disorders such as neuropathic pain and epilepsy. Similarly, a more specific therapeutic agent for NKCC1b compared to NKCC1a would be more effective in treating such disorders.

  The methods of the invention include, for example, the following etiologies: alcohol abuse; diabetes; eosinophil myalgia syndrome; Gann-Barre syndrome; exposure to heavy metals such as arsenic, lead, mercury and thallium; HIV / AIDS; / Exposure to AIDS drugs; malignant tumors; pharmaceuticals such as amiodarone, aurothioglucose, cisplatinum, dapsone, stavudine, zalcitabine, didanosine, disulfiram, FK506, hydrazine, isoniazid, metronidazole, nitrofurantoin, paclitaxel, phenytoin and vincristine; Monoclonal gammopathy; multiple sclerosis; post-stroke central pain; postherpetic neuralgia; trauma such as carpal tunnel syndrome, cervical or lumbar radiculopathy, complex focal pain syndrome, spinal cord injury and blocking neuralgia Trigeminal neuralgia; vasculitis Vitamin B6 large doses; and specific vitamin deficiency (B12, B1, B6, E) may be used for the treatment and / or prophylaxis of neuropathic pain with. Neuropsychiatric disorders that are effectively treated using the methods of the present invention include, but are not limited to, bipolar disorder, anxiety disorder, panic disorder, depression, schizophrenia, obsessive compulsive disorder and post-traumatic stress syndrome.

Effectively use compositions in the method of the present invention ^Na + ^-K + -2Cl ^- reducing the effective amount of (NKCC) symporter, inactivating, and / or inhibits the activity be able to. Preferably, such compositions are capable of reducing, inactivating, and / or inhibiting activity of the cotransporter NKCC1. In certain embodiments, a composition of the invention comprises an NKCC1 antagonist (eg, but not limited to a small molecule inhibitor of NKCC1, an antibody or antigen-binding fragment thereof that specifically binds NKCC1, and a soluble NKCC1 ligand); An antisense oligonucleotide; an NKCC1-specific short interfering RNA molecule (siRNA or RNAi); and at least one therapeutic agent selected from the group consisting of engineered soluble NKCC1 molecules. In preferred embodiments, the therapeutic agent is a CNS targeting NKCC cotransporter antagonist such as furosemide, bumetanide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide, tripamide, etc .; thiazide and thiazide-like diuretics such as bendroflumethiazide, benz Thiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylcrothiazide, polythiazide, trichloromethiazide, chlorothalidon, indapamide, metrazone and kinetazone; and analogs and functional derivatives of such compounds.

  Analogs of CNS targeting NKCC cotransporter antagonists that may be used in the methods of the present invention are those of formulas I, II and / or III

［式中、
Ｒ_１は存在しないか、Ｈ又はＯであり；
Ｒ_２はＨであるか、又は、Ｒ_１がＯである場合は、アルキルアミノジアルキル、アルキルアミノカルボニルジアルキル、アルキルオキシカルボニルアルキル、アルキルアルデヒド、アルキルケトンアルキル、アルキルアミド、アルキルアンモニウム基、アルキルカルボン酸、アルキルヘテロアリール、アルキルヒドロキシ、生体適合性重合体、例えばアルキルオキシ（ポリアルキルオキシ）アルキルヒドロキシ、ポリエチレングリコール（ＰＥＧ）、ポリエチレングリコールエステル（ＰＥＧエステル）、ポリエチレングリコールエーテル（ＰＥＧエーテル）、メチルオキシアルキル、メチルオキシアルカリル、メチルチオアルキルアルキル及びメチルチオアルカリルの非置換又は置換されたものからなる群より選択され、
そしてＲ_１が存在しない場合は、Ｒ_２は水素、ジアルキルアミノ、ジアリールアミノ、ジアルキルアミノジアルキル、ジアルキルカルボニルアミノジアルキル、ジアルキルエステルアルキル、ジアルキルアルデヒド、ジアルキルケトンアルキル、ジアルキルアミド、ジアルキルカルボン酸及びジアルキルヘテロアリールの非置換又は置換されたものからなる群より選択され；
Ｒ_３はアリール、ハロ、ヒドロキシ、アルコキシ及びアリールオキシの非置換又は置換されたものからなる群より選択され；
Ｒ_４及びＲ_５は各々独立して水素、アルキルアミノジアルキル、アルキルヒドロキシアミノジアルキルの非置換又は置換されたものからなる群より選択される］の化合物又はその薬学的に受容可能な塩、溶媒和物、互変異性体又は水和物を包含する。

[Where:
R ₁ is absent, H or O;
R ₂ is H, or when R ₁ is O, alkylaminodialkyl, alkylaminocarbonyldialkyl, alkyloxycarbonylalkyl, alkylaldehyde, alkylketone alkyl, alkylamide, alkylammonium group, alkylcarboxylic acid Alkyl heteroaryl, alkyl hydroxy, biocompatible polymers such as alkyloxy (polyalkyloxy) alkyl hydroxy, polyethylene glycol (PEG), polyethylene glycol ester (PEG ester), polyethylene glycol ether (PEG ether), methyloxyalkyl Selected from the group consisting of unsubstituted or substituted methyloxyalkaryl, methylthioalkylalkyl and methylthioalkaryl.
And if R ₁ is not present, R ₂ is hydrogen, dialkylamino, diarylamino, dialkylaminodialkyl, dialkylcarbonylaminodialkyl, dialkyl ester alkyl, dialkyl aldehyde, dialkyl ketone alkyl, dialkylamide, dialkylcarboxylic acid and dialkylheteroaryl Selected from the group consisting of unsubstituted or substituted
R ₃ is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy unsubstituted or substituted;
R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, and unsubstituted or substituted alkylhydroxyaminodialkyl] or a pharmaceutically acceptable salt or solvate thereof Product, tautomer or hydrate.

In some embodiments of the invention, the analog is bumetanide aldehyde, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ester, bumetanide 3- (dimethylaminopropyl) ester, bumetanide N, N-diethyl glycolamide ester, bumetanide dimethylglycolamide ester, bumetanide pibaxetyl ester, bumetanide methoxy (polyethyleneoxy) _n-1 ethyl ester, bumetanide benzyltrimethylammonium salt, and bumetanide cetyl It can be a trimethylammonium salt.

According to another embodiment of the invention, the analog is furosemide aldehyde, furosemide ethyl ester, furosemide cyanomethyl ester, furosemide benzyl ester, furosemide morpholinoethyl ester, furosemide 3- (dimethylaminopropyl) ester, furosemide N, N-diethyl. Glycolamide ester, furosemide dibenzylamide, furosemide benzylmethylammonium salt, furosemide cetyltrimethylammonium salt, furosemide N, N-dimethylglycolamide ester, furosemide methoxy (polyethyleneoxy) _n-1 ethyl ester, furosemide pibaxetyl ester, and It can be a furosemide propaxetyl ester.

In yet another embodiment of the invention, the analog is piretanide aldehyde, piretanide methyl ester, piretanide cyanomethyl ester, piretanide benzyl ester, piretanide morpholinoethyl ester, piretanide 3- (dimethylamino) Propyl) ester, Piretanide N, N-diethylglycolamide ester, Piretanide diethylamide, Piretanide dibenzylamide, Piretanide benzyltrimethylammonium salt, Piretanide cetyltrimethylammonium salt, Piretanide N, N-dimethylglycolamide ester , Piretanide methoxy (polyethyleneoxy) _n-1 ethyl ester, piretanide pibaxetyl ester, and piretanide propaxetyl ester.

  Analogs of CNS targeting NKCC cotransporter antagonists usefully used in the methods of the invention have the following formula IV:

［式中、
Ｒ_３、Ｒ_４及びＲ_５は上記の通り定義され；そして、
Ｒ_６はアルキルオキシカルボニルアルキル、アルキルアミノカルボニルジアルキル、アルキルアミノジアルキル、アルキルヒドロキシ、生体適合性重合体、例えばアルキルオキシ（ポリアルキルオキシ）アルキルヒドロキシ、ポリエチレングリコール（ＰＥＧ）、ポリエチレングリコールエステル（ＰＥＧエステル）、ポリエチレングリコールエーテル（ＰＥＧエーテル）、メチルオキシアルキル、メチルオキシアルカリル、メチルチオアルキル及びメチルチオアルカリルの非置換又は置換されたものからなる群より選択される］の化合物又はその薬学的に受容可能な塩、溶媒和物、互変異性体又は水和物を包含する。

[Where:
R ₃ , R ₄ and R ₅ are defined as above; and
R ₆ is alkyloxycarbonylalkyl, alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, biocompatible polymer such as alkyloxy (polyalkyloxy) alkylhydroxy, polyethylene glycol (PEG), polyethylene glycol ester (PEG ester) Selected from the group consisting of unsubstituted or substituted ones of polyethylene glycol ether (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl] or a pharmaceutically acceptable compound thereof Includes salts, solvates, tautomers or hydrates.

  In some embodiments of the invention, the analog is a tetrazolyl substituted azosemide (eg, methoxymethyltetrazolyl substituted azosemide, methylthiomethyltetrazolyl substituted azosemide and N-mPEG350-tetrazolyl substituted azosemide), an azosemide benzyltrimethylammonium salt. And / or may be selected from the group consisting of azosemidocetyltrimethylammonium salts.

  Analogs usefully used in the method of the present invention are represented by the following formula V:

［式中、
Ｒ_７はアルキルオキシカルボニルアルキル、アルキルアミノカルボニルジアルキル、アルキルアミノジアルキル、アルキルヒドロキシ、生体適合性重合体、例えばアルキルオキシ（ポリアルキルオキシ）アルキルヒドロキシ、ポリエチレングリコール（ＰＥＧ）、ポリエチレングリコールエステル（ＰＥＧエステル）、ポリエチレングリコールエーテル（ＰＥＧエーテル）、メチルオキシアルキル、メチルオキシアルカリル、メチルチオアルキル及びメチルチオアルカリルの非置換又は置換されたものからなる群より選択され；そして、
Ｘ⁻はハライド、例えばブロミド、クロリド、フロリド、ヨーダイド、又は、アニオン性部分、例えばメシレート、トシレートであるか；或いは、Ｘ⁻は存在せず、そして化合物はスルホニル尿素部分（−ＳＯ_２−ＮＨ−ＣＯ−）からプロトンを消失することにより「内部」又は両性イオン性の塩を形成する］の化合物又はその薬学的に受容可能な塩、溶媒和物、互変異性体又は水和物を包含する。

[Where:
R ₇ is alkyloxycarbonylalkyl, alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, biocompatible polymer such as alkyloxy (polyalkyloxy) alkylhydroxy, polyethylene glycol (PEG), polyethylene glycol ester (PEG ester) Selected from the group consisting of unsubstituted or substituted polyethylene glycol ether (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl; and
X ⁻ is a halide such as bromide, chloride, fluoride, iodide, or an anionic moiety such as mesylate, tosylate; or X ⁻ is absent and the compound is a sulfonylurea moiety (—SO ₂ —NH— Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, which forms an “internal” or zwitterionic salt by eliminating protons from CO−) .

  In some embodiments of the invention, the analog may be selected from the group consisting of a pyridine substituted torsemide quaternary ammonium salt or the corresponding internal salt (zwitterion). Examples include, but are not limited to, methoxymethylpyridinium torsemide salt, ocular hillthiomethylpyridinium torsemide salt and N-mPEG350-pyridinium torsemide salt.

  Any of the R groups defined herein can be excluded from the analogs disclosed herein.

  Intermediate compounds formed through the synthetic methods described below to obtain compounds of formula I, II, III, IV and / or V are also useful as therapeutic agents for neuropsychiatric disorders as described herein May have sex.

  Modifications of CNS targeting NKCC cotransporter antagonists used in the methods of the present invention may include functional groups and / or aluminum hydrides, alkyl halides, alcohols, aldehydes, aryl halides, alkylamides, arylamines and quaternary ammonium salts. It can include reacting an antagonist with a compound selected from the group consisting of unsubstituted or substituted or combinations thereof. Non-limiting examples of compounds that can be used as starting materials are illustrated below.

「アルキル」という用語は本明細書においては、直鎖又は分枝鎖の飽和又は部分不飽和の炭化水素基を指す。アルキル基の例は限定しないがメチル、エチル、イソプロピル、ｔ−ブチル、ｎ−ペンチル等を包含する。「不飽和」とは１、２又は３つの二重結合又は三重結合又はその組合せの存在を意味する。そのようなアルキル基は場合により後述するとおり置換され得る。

The term “alkyl” as used herein refers to a straight or branched chain saturated or partially unsaturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, t-butyl, n-pentyl and the like. “Unsaturated” means the presence of 1, 2 or 3 double bonds or triple bonds or combinations thereof. Such alkyl groups can be optionally substituted as described below.

  The term “alkylene” as used herein refers to a straight chain having two central terminal monovalent radicals derived by removing one hydrogen atom from each of the two terminal carbon atoms of the linear parent alkane. Or it refers to a branched chain.

  The term “aryl” as used herein refers to an aromatic group, or optionally suitable substituents such as, but not limited to, lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl. , Oxo, hydroxy, mercapto, optionally substituted amino, carboxy, tetrazolyl, optionally substituted carbamoyl optionally substituted with alkyl, optionally substituted aminosulfonyl, acyl, aroyl, heteroaryl, acyloxy, aroyloxy, Heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, or optionally substituted aromatic group when fused to one or more of the optionally substituted aromatic groups. In is also possible multiple substitutions. Examples of aryl include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl and the like.

  The term “halo” as used herein refers to bromo, chloro, fluoro or iodo. Alternatively, the term “halide” as used herein refers to bromide, chloride, fluoride or iodide.

  The term “hydroxy” as used herein refers to the group —OH.

  The term “alkoxy” as used herein, alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, t-butoxy, pentyloxy, hexyloxy and the like.

  The term “aryloxy” as used herein refers to the group —ArO, where Ar is aryl or heteroaryl. Examples include but are not limited to phenoxy, benzyloxy and 2-naphthyloxy.

The term “amino” as used herein refers to —NH ₂ where one or both of the hydrogen atoms may be replaced by optionally one of alkyl or aryl or each optionally substituted.

  The term “alkylthio” as used herein, alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfur moiety. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, and the like.

The term “carboxy” as used herein refers to the group —CO ₂ H.

The term “quaternary ammonium” as used herein refers to a chemical structure having four bonds to a positively charged nitrogen on a nitrogen in the “onium” state, ie, “R4N ⁺ ” or “quaternary nitrogen”. Where R is an organic substituent such as alkyl or aryl. The term “quaternary ammonium salt” as used herein refers to the association of a quaternary ammonium with a cation.

  The term “substituted” as used herein refers to the replacement of one or more hydrogen atoms of a group that is replaced by substituents known to those skilled in the art and results in the stable compounds described below. Examples of suitable replacement groups include, but are not limited to, alkyl, acyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, acyl, amino, amide, carboxy, carboxyalkyl, carboxyaryl, halo, oxo, Mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, dialkoxymethyl, cycla, heterocycloalkyl, dialkylaminoalkyl, carboxylic acid, carboxyamide, haloalkyl, alkylthio, aralkyl, alkylsulfonyl, arylthio, amino, alkylamino, dialkyl Includes amino, guanidino, ureido and the like. Substitution is permitted when such a combination results in a compound that is stable for the intended use. For example, substitutions are permitted when the resulting compound is isolated from the reaction mixture to a useful degree of purity and is robust enough to withstand formulation into a therapeutic or diagnostic agent. .

  The term “solvate” is used herein to refer to a compound that retains the biological effectiveness of the compound resulting from the physical association of the particular compound with, for example, one or more solvent molecules. Refers to an acceptable solvated form. Examples of solvates include, but are not limited to, compounds of the present invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid or ethanolamine.

  The term “hydrate” as used herein refers to a compound where the solvent is water.

The term “biocompatible polymer” as used herein refers to a polymer moiety that is substantially non-toxic and does not tend to produce a substantial immune response, clotting or other undesirable effects. . Polyalkylene glycol is a biocompatible polymer, where polyalkylene glycol is a linear or branched polyalkylene glycol polymer such as polyethylene glycol, polypropylene glycol and polybutylene glycol. And further includes monoalkyl ethers of polyalkylene glycols. In some embodiments of the invention, the polyalkylene glycol polymer is a lower alkyl polyalkylene glycol moiety, such as a polyethylene glycol moiety (PEG), a polypropylene glycol moiety or a polybutylene glycol moiety. PEG has the formula _{HO (CH 2 CH 2 O)} n H, wherein n can be from about 1 to about 4000 or more. In some embodiments, n is 1-100, and in other embodiments n is 5-30. The PEG moiety can be linear or branched. In another embodiment, PEG can be attached to a group such as hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, the PEG can be an alkoxy PEG, such as methoxy-PEG (or mPEG), where one end is a relatively inert alkoxy group and the other end is a hydroxyl group. is there.

  Compounds of formula I, II, III, IV and / or V can be synthesized using traditional synthetic techniques well known in the art. The more specific synthesis route is as described below.

  Bumetanide analogs are synthesized by reacting the carboxylic acid moiety of bumetanide with various reagents. For example, bumetanide may undergo esterification via reaction with an alcohol, such as a linear, branched, substituted or unsubstituted alcohol. Bumetanide is also alkylated via reaction with suitable substituted or unsubstituted alkyl and aryl halides such as chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, N-diethylacetamide and the like. Can be PEG type esters can be obtained by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or alkyloxy (polyalkyloxy) alkyl tosylate such as MeO-PEG1000-OTs. Can be formed. Esters of the “Axetil” type can also be formed by alkylation with alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Bumetanide can also be converted to an appropriate substituted or unsubstituted alkylamine after conversion to acid chloride or by use of an activator such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Alternatively, amidation may be caused by reaction with arylamine. Bumetanide may also react with quaternary ammonium hydroxides such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide to form bumetanide quaternary ammonium salts. Schemes 1 and 7 below show synthetic schemes for some exemplary compounds of Formula I.

  Scheme 1. Synthesis of exemplary compounds according to Formula I

フロセミドアナログはブメタニドアナログの合成に使用したものと同様の方法によって合成する。特に、フロセミドはアルコール、例えば直鎖、分枝鎖、置換又は非置換のアルコールとの反応を介してエステル化を起こす場合がある。フロセミドは又適当な置換又は非置換のアルキルハライド及びアリールハライド、例えば、クロロアセトニトリル、ベンジルクロリド、１−（ジメチルアミノ）プロピルクロリド、２−クロロ−Ｎ，Ｎ−ジエチルアセトアミド等との反応を介してアルキル化し得る。ＰＥＧ型のエステルはＭｅＯ−ＰＥＧ３５０−Ｃｌ等のようなアルキルオキシ（ポリアルキルオキシ）アルキルハライド又はＭｅＯ−ＰＥＧ１０００−ＯＴｓ等のようなアルコルオキシ（ポリアルキルオキシ）アルキルトシレートを用いたアルキル化により形成し得る。「アキセチル」型のエステルもまた、アルキルハライド、例えばクロロメチルピバリレート又はクロロメチルプロピオネートを用いたアルキル化により形成し得る。フロセミドはまた、酸クロリドへの変換の後、又は、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）のような活性化剤の使用により、適当な置換又は非置換のアルキルアミン又はアリールアミンとの反応によりアミド化を起こす場合がある。フロセミドは又、四級アンモニウム水酸化物、例えばベンジルトリメチルアンモニウム水酸化物又はセチルトリメチルアンモニウム水酸化物と反応して、フロセミド四級アンモニウム塩を形成する場合がある。以下のスキーム２は式ＩＩの一部の例示的化合物の合成スキームを示す。

Furosemide analogs are synthesized by a method similar to that used for the synthesis of bumetanide analogs. In particular, furosemide may undergo esterification via reaction with alcohols, such as linear, branched, substituted or unsubstituted alcohols. Furosemide can also be reacted through reaction with suitable substituted or unsubstituted alkyl and aryl halides such as chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, N-diethylacetamide and the like. Can be alkylated. PEG-type esters are formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or the like, or alcoholyl (polyalkyloxy) alkyl tosylate such as MeO-PEG1000-OTs. Can do. “Axetyl” type esters can also be formed by alkylation with alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Furosemide can also be converted to the appropriate substituted or unsubstituted alkylamine after conversion to acid chloride or by the use of an activator such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Alternatively, amidation may be caused by reaction with arylamine. Furosemide may also react with quaternary ammonium hydroxides such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide to form furosemide quaternary ammonium salts. Scheme 2 below shows a synthetic scheme for some exemplary compounds of Formula II.

  Scheme 2. Synthesis of exemplary compounds according to Formula II

ピレタニドアナログはブメタニドアナログの合成に使用したものと同様の方法によって合成する。特に、ピレタニドはアルコール、例えば直鎖、分枝鎖、置換又は非置換のアルコールとの反応を介してエステル化を起こす場合がある。ピレタニドは又、適当な置換又は非置換のアルキルハライド又はアリールハライド例えば、クロロアセトニトリル、ベンジルクロリド、１−（ジメチルアミノ）プロピルクロリド、２−クロロ−Ｎ，Ｎ−ジエチルアセトアミド等との反応を介してアルキル化し得る。ＰＥＧ型のエステルはＭｅＯ−ＰＥＧ３５０−Ｃｌ等のようなアルキルオキシ（ポリアルキルオキシ）アルキルハライド又はＭｅＯ−ＰＥＧ１０００−ＯＴｓ等のようなアルコルオキシ（ポリアルキルオキシ）アルキルトシレートを用いたアルキル化により形成し得る。「アキセチル」型のエステルもまた、アルキルハライド、例えばクロロメチルピバリレート又はクロロメチルプロピオネートを用いたアルキル化により形成し得る。ピレタニドはまた、酸クロリドへの変換の後、又は、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）のような活性化剤の使用により、適当な置換又は非置換のアルキルアミン又はアリールアミンとの反応によりアミド化を起こす場合がある。ピレタニドは又、四級アンモニウム水酸化物、例えばベンジルトリメチルアンモニウム水酸化物又はセチルトリメチルアンモニウム水酸化物と反応して、ピレタニド四級アンモニウム塩を形成する場合がある。以下のスキーム３は式ＩＩＩの一部の例示的化合物の合成スキームを示す。

Piretanide analogs are synthesized by a method similar to that used for the synthesis of bumetanide analogs. In particular, piretanide may undergo esterification via reaction with alcohols such as linear, branched, substituted or unsubstituted alcohols. Piretanide can also be reacted through reaction with a suitable substituted or unsubstituted alkyl halide or aryl halide such as chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, N-diethylacetamide and the like. Can be alkylated. PEG-type esters are formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or the like, or alcoholyl (polyalkyloxy) alkyl tosylate such as MeO-PEG1000-OTs. Can do. “Axetyl” type esters can also be formed by alkylation with alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Piretanides can also be used in suitable substituted or unsubstituted alkylamines after conversion to acid chloride or by use of an activator such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Alternatively, amidation may occur by reaction with arylamine. Piretanide may also react with quaternary ammonium hydroxides, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form piretanide quaternary ammonium salts. Scheme 3 below shows a synthetic scheme for some exemplary compounds of Formula III.

  Scheme 3. Synthesis of exemplary compounds according to Formula III

アゾセミドアナログは種々の試薬にアゾセミドのテトラゾリル部分を反応させることにより合成する。アゾセミドはアルデヒドの添加によってヒドロキシアルキル化を起こす場合があり、これによってヒドロキシアルキル基が形成される。更に、アルコールをアルデヒドと反応させ、エーテルを得うる。更に、アルキルチオールをアルデヒドとともに添加し、チオールエーテルを形成し得る。アゾセミドは又適当なアルキルハライド又はアリールハライド、例えばメチルクロロメチルエステル及びベンジルクロロメチルチオエーテルのようなエーテル又はチオエーテル結合を有するアルキル又はアリールハライドを添加することによりアルキル化し得る。ＰＥＧ型のエステルはＭｅＯ−ＰＥＧ３５０−Ｃｌ等のようなアルキルオキシ（ポリアルキルオキシ）アルキルハライド又はＭｅＯ−ＰＥＧ１０００−ＯＴｓ等のようなアルコルオキシ（ポリアルキルオキシ）アルキルトシレートを用いたアルキル化により形成し得る。「アキセチル」型のエステルもまた、アルキルハライド、例えばクロロメチルピバリレート又はクロロメチルプロピオネートを用いたアルキル化により形成し得る。アゾセミドはアゾセミド四級アンモニウム塩を形成するために、四級アンモニウム塩、例えばベンジルトリメチルアンモニウム臭化物及び水酸化ナトリウムのような塩基、又はセチルトリメチルアンモニウム及び水酸化ナトリウムのような塩基と反応させ得る。以下のスキーム４は式ＩＶの一部の例示的化合物の合成スキームを示す。

Azosemide analogs are synthesized by reacting various reagents with the tetrazolyl moiety of azosemide. Azosemides can undergo hydroxyalkylation by the addition of aldehydes, thereby forming hydroxyalkyl groups. Furthermore, an alcohol can be reacted with an aldehyde to obtain an ether. In addition, alkyl thiols can be added with aldehydes to form thiol ethers. Azosemides can also be alkylated by adding suitable alkyl or aryl halides, such as alkyl or aryl halides having ether or thioether linkages such as methyl chloromethyl ester and benzyl chloromethyl thioether. PEG-type esters are formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or the like, or alcoholyl (polyalkyloxy) alkyl tosylate such as MeO-PEG1000-OTs. Can do. “Axetyl” type esters can also be formed by alkylation with alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Azosemide can be reacted with bases such as quaternary ammonium salts such as benzyltrimethylammonium bromide and sodium hydroxide, or bases such as cetyltrimethylammonium and sodium hydroxide to form azosemide quaternary ammonium salts. Scheme 4 below shows a synthetic scheme for some exemplary compounds of Formula IV.

  Scheme 4. Synthesis of exemplary compounds according to Formula IV

トルセミド（トラセミドとしても知られている）アナログは種々の試薬にトルセミドを反応させることにより合成する。Ｎ−置換四級アンモニウム塩を形成するために、トルセミドは適当なアルキル又はアリールハライド、例えばベンジルクロリドの添加によってアルキル化を起こす場合がある。Ｎ−置換エーテル四級アンモニウム塩を形成するために、エーテル結合を有するアルキルハライド及びアリールハライド、例えばメチルクロロメチルエーテル及びベンジルクロロメチルエーテルを使用し得る。Ｎ−置換チオエーテル四級アンモニウム塩を形成するために、チオエーテル結合を有するアルキルハライド及びアリールハライド、例えばメチルクロロメチルチオエーテル及びベンジルクロロメチルチオエーテルを使用し得る。ＰＥＧ型のエーテル含有四級アンモニウム塩はＭｅＯ−ＰＥＧ３５０−Ｃｌ等のようなアルキルオキシ（ポリアルキルオキシ）アルキルハライド又はＭｅＯ−ＰＥＧ１０００−ＯＴｓ等のようなアルコルオキシ（ポリアルキルオキシ）アルキルトシレートを用いたアルキル化により形成し得る。「アキセチル」型の四級アンモニウム塩もまた、アルキルハライド、例えばクロロメチルピバリレート又はクロロメチルプロピオネートの添加を介して形成し得る。以下のスキーム５は式Ｖの一部の例示的化合物の合成スキームを示す。

Torsemide (also known as torasemide) analogs are synthesized by reacting torsemide with various reagents. In order to form N-substituted quaternary ammonium salts, torsemide may undergo alkylation by addition of a suitable alkyl or aryl halide such as benzyl chloride. Alkyl halides and aryl halides with ether linkages such as methyl chloromethyl ether and benzyl chloromethyl ether may be used to form N-substituted ether quaternary ammonium salts. To form N-substituted thioether quaternary ammonium salts, alkyl and aryl halides with thioether linkages such as methyl chloromethyl thioether and benzyl chloromethyl thioether can be used. PEG type ether-containing quaternary ammonium salts use alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or alcohol oxy (polyalkyloxy) alkyl tosylate such as MeO-PEG1000-OTs. Can be formed by conventional alkylation. “Axetyl” type quaternary ammonium salts may also be formed through the addition of alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Scheme 5 below shows a synthetic scheme for some exemplary compounds of Formula V.

  Scheme 5. Synthesis of exemplary compounds according to Formula V

置換安息香酸ブメタニド、ピレタニド及びフロセミドは、文献の方法によりアミン置換アンモニウムハイドライド、例えばビス（４−メチルピペラジニル）アルミニウムハイドライドを用いて対応するブメタニドアルデヒド、ピレタニドアルデヒド及びフロセミドアルデヒドに選択的に還元できる。Ｍｕｒａｋｉ，Ｍ．及びＭｕｋｉａｙａｍａ，Ｔ．，Ｃｈｅｍ．Ｌｅｔｔｅｒｓ，１９７４，１４４７；Ｍｕｒａｋｉ，Ｍ．及びＭｕｋｉａｙａｍａ，Ｔ．，Ｃｈｅｍ．Ｌｅｔｔｅｒｓ，１９７５，２１５；及びＨｕｂｅｒｔ，Ｔ．等，Ｊ．Ｏｒｇ．Ｃｈｅｍ．，１９８４，２２７９を参照される。より親油性のベンズアルデヒドはより親油性の安息香酸に容易に大気酸化し、ベンズアルデヒドも又、ＮＡＤＰＨコファクターの使用を介し、数種の酸化Ｐ４５０酵素とともに生体内で対応する安息香酸に代謝されることがよく知られている。

Substituted bumetanide benzoate, pyrethanide and furosemide are selected for the corresponding bumetanide aldehyde, piretanide aldehyde and furosemide aldehyde using amine substituted ammonium hydride, eg bis (4-methylpiperazinyl) aluminum hydride, according to literature methods Can be reduced. Muraki, M .; And Mukiyama, T .; , Chem. Letters, 1974, 1447; Muraki, M .; And Mukiyama, T .; , Chem. Letters, 1975, 215; and Hubert, T .; J. et al. Org. Chem. , 1984, 2279. More lipophilic benzaldehyde is easily oxidized to the more lipophilic benzoic acid in the atmosphere, and benzaldehyde is also metabolized in vivo to the corresponding benzoic acid with several oxidized P450 enzymes through the use of NADPH cofactor. Is well known.

  Scheme 6. Synthesis of exemplary bumetanide, piretanide and furosemide benzaldehyde analogs

安息香酸から対応するベンズアルデヒドに変換するために使用した還元操作法については、Ｍｕｒａｋｉ，Ｍ．及びＭｕｋｉａｙａｍａ，Ｔ．，Ｃｈｅｍ．Ｌｅｔｔｅｒｓ，１９７４，１４４７；同書，１９７５，２１５；Ｈｕｂｅｒｔ，Ｔ．Ｄ．，Ｅｙｍａｎ，Ｄ．Ｐ．及びＷｉｅｍｅｒ，Ｄ．Ｆ．，Ｊ．Ｏｒｇ．Ｃｈｅｍ．，１９８４，２２７９を参照
スキーム７．例示的なブメタニド、ピレタニド及びフロセミドのポリエチレングリコールエステルの合成

For the reduction procedure used to convert benzoic acid to the corresponding benzaldehyde, see Muraki, M .; And Mukiyama, T .; , Chem. Letters, 1974, 1447; ibid., 1975, 215; Hubert, T .; D. Eyman, D .; P. And Wiemer, D .; F. , J .; Org. Chem. , 1984, 2279. Scheme 7. Synthesis of exemplary bumetanide, piretanide and furosemide polyethylene glycol esters

ＰＥＧ−ＸはＸ−（ＣＨ_２）_ｍ（ＯＣＨ_２ＣＨ_２）_ｎ−１−Ｙであり、ここでＸはハロ又は他の脱離基（メシレート「ＯＭｓ」、トシレート「ＯＴｓ」）及びＹはＯＨ又はアルコール保護基、例えばアルキル基、アリール基、アシル基又はエステル基であり、そしてここでｍ＝１−５及びｎ＝１−１００である。

PEG-X is _{X- (CH 2) m (OCH} 2 CH 2) n-1 -Y, where X is halo or other leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group, an aryl group, an acyl group or an ester group, where m = 1-5 and n = 1-100.

  Scheme 8. Synthesis of exemplary azosemide and torsemide alkyl polyethylene glycol ethers

ＰＥＧ−ＸはＸ−（ＣＨ_２）_ｍ（ＯＣＨ_２ＣＨ_２）_ｎ−１−Ｙであり、ここでＸはハロ又は他の脱離基（メシレート「ＯＭｓ」、トシレート「ＯＴｓ」）及びＹはＯＨ又はアルコール保護基、例えばアルキル基、アリール基、アシル基又はエステル基であり、そしてここでｍ＝１−５及びｎ＝１−１００である。

PEG-X is _{X- (CH 2) m (OCH} 2 CH 2) n-1 -Y, where X is halo or other leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group, an aryl group, an acyl group or an ester group, where m = 1-5 and n = 1-100.

  Starting materials for synthesizing the compounds of the present invention are further described in US Pat. No. 3,634,583 to Feit; US Pat. No. 3,806,534 to Fiet; US Pat. No. 3,058,882 to Struem et al .; US to Bormann Patents 4,010,273; U.S. Pat. No. 3,665,002 to Popelak; and U.S. Pat. No. 3,665,002 to Delarge, the disclosure of which is hereby incorporated by reference.

  The compounds of the present invention can contain isomers, tautomers, zwitterions, enantiomers, diastereomers, racemic mixtures or stereochemical mixtures thereof. The term “isomer” as used herein refers to compounds having the same number and type of atoms, and thus the same molecular weight, but differing in terms of atomic arrangement or arrangement in space. Furthermore, the term “isomer” includes stereoisomers and geometric isomers. The terms “stereoisomer” or “optical isomer” as used herein have limited rotation caused by at least one chiral atom or vertical asymmetric surface (eg, certain biphenyl, allene, and spiro compounds) Represents a stable isomer capable of rotating surface polarized light. The present invention contemplates stereoisomers and mixtures thereof because asymmetric centers and other chemical structures can exist in some compounds of the present invention that can give rise to stereoisomers. The compounds of the invention and their salts can contain asymmetric carbon atoms and can therefore exist as monostereoisomers, racemic mixtures and enantiomers, and mixtures of diastereomers. Typically such compounds are prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated as pure stereoisomers, for example as independent enantiomers or diastereomers, or as stereoisomer-enriched mixtures. Tautomers can easily be converted internally to native isomers, with altered ligand connectivity, such as the keto and enol forms of acetoacetate ether. The inventive methods and compositions employ tautomers of the aforementioned compounds. Zwitterions are internal salts or dipolar compounds that possess acidic and basic groups in the same molecule. In neutral PH, most zwitterion cations and anions are equally ionized.

  The present invention further provides prodrugs having the compounds described herein. The term “prodrug” is intended to denote a compound that is converted to a specified compound that is pharmaceutically / pharmacologically active by solvolysis or under metabolically physiological conditions. A prodrug may be a chemically derived compound of the invention, for example: (i) retains, retains, or does not retain at all, the biological activity of the parent drug compound, and (ii) the parent Metabolized to obtain a chemical compound. The prodrugs of the present invention may also be “partial prodrugs” which are chemically derived compounds, for example: (i) retain some or all of the biological activity of the parent drug compound. Or (ii) metabolized to obtain biologically active derivatives of the compound. Prodrugs can be formed utilizing hydrolyzable couplings to the compounds described herein. For further discussion on prodrugs, see Ettmayer et al. Med. Chem. 47 (10): 2394-2404 (2004).

  The prodrugs of the present invention can cross the blood brain barrier and can be hydrolyzed by CNS esterase to provide the active compound. In addition, the prodrugs provided herein also have improved bioavailability, improved water solubility, improved passive intestinal absorption, improved delivery media intestinal absorption, protection against accelerated metabolism, against target tissues Tissue selective delivery and / or passive enhancement may also be shown.

  The prodrugs of the present invention can contain compounds according to Formulas 1, II, III, IV and / or V described herein. The prodrugs of the present invention are not only similar derivatives of indacrinone and ozolinone, but also bumetanide, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ether, bumetanide 3- (dimethylaminopropyl) ester, bumetanide N, N-diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide pibaxetyl ester, furosemide, furosemide ethyl ester, furosemide cyanomethyl ester, furosemide benzyl ester, furosemide morpholinoethyl ester, furosemide 3- (dimethyl Aminopropyl) ester, furosemide N, N-diethylglycolamide ester, furosemide dibenzylamide, furosemide benzyltrimethyl-ammonium salt Furosemide cetyl trimethyl ammonium salt, Furosemide N, N-dimethylglycolamide ester, Furosemide pibaxetyl ester, Furosemide propaxetyl ester, Piretanide, Piretanide methyl ester, Piretanide cyanomethyl ester, Piretanide benzyl ester, Pirete Tanide morpholino ethyl ester, Piretanide 3- (dimethylaminopropyl) ester, Piretanide N, N-diethyl glycolamide ester, Piretanide diethylamide, Piretanide dibenzylamide, Piretanide benzyltrimethylammonium salt, Piretanide cetyltrimethyl Ammonium salt, Piretanide N, N-dimethylglycolamide ester, Piretanide pibaxetyl ester, Piretanide propaxetyl ester, Tetrazoli Substituted Azosemide, containing pyridinium substituted Torsemide salt (also known as pyrimidine substituted torsemide quaternary ammonium salt). See the scheme shown earlier.

  Furthermore, attaching a biocompatible polymer as described above, such as polyethylene glycol (PEG), to a compound of the invention using a bond that degrades under physiological conditions, as described in the scheme shown previously. Can form a prodrug. Schacht, E .; H. etc. See also Poly (ethylene glycol) Chemistry and Biological Applications, American Chemical Society, San Francisco, CA 297-315 (1997). Attachment of PEG to proteins can be used to reduce the immunogenicity and / or increase half-life of the compounds provided herein. Any conventional PEGylation method can be used as long as the PEGylating agent retains pharmacological activity.

  The composition of the requirements invention is suitable for human and livestock applications and is preferably delivered as a pharmaceutical composition. A pharmaceutical composition consists of one or more therapeutic agents, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable salt refers to a salt form of a compound that is allowed to be used or manufactured as a pharmaceutical, which retains the biological effectiveness of the free acid and base of the particular compound, It is not biologically or otherwise undesirable. Examples of such salts are: Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wermuth, C .; G. And Stahl, P .; H. (Eds.), Wiley-Verlag Helvetica Acta, Zurich, 2002 [ISBN3-906390-26-8]. Examples of such salts include alkali metal salts and additional salts of free acids and bases. Examples of pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, phosphorus dihydrogen Salt, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate , Heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-diionate, hexyne-1 , 6-Dionate, Benzoate, Chlorobenzoate, Methylbenzoate, Dinitrobenzoate, Hydroxybenzoate, Methoxybenzoate, Phthalate, Xylenesulfonate, Phenylacetate, Phenylpropionate On acid, fu Nylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, ethanesulfonate, propanesulfonate, toluenesulfonate, naphthalene-1-sulfonic acid Contains salts, naphthalene-2-sulfonate and mandelate.

  The pharmaceutical composition of the present invention may also contain other compounds, which may be physiologically active or inactive. For example, one or more therapeutic agents of the present invention can be treated according to the usage of the present invention with the addition of other agents in a therapeutic combination. Such combinations can be treated as separate compositions and combined to deliver in a complementary delivery system, or form a combined composition, such as a mixture or a fusion compound. In addition, the combination of treatments described above may contain a BBB permeability enhancer and / or a penetrating agent.

  Carriers and additives using such pharmaceutical compositions can take a variety of forms depending on the desired method of administration. Accordingly, compositions for oral administration include tablets, dragees, hard capsules, soft capsules using appropriate carriers and additives such as starches, sugars, binders, diluents, granulating agents, lubricants, tablet disintegrating agents and the like. , Solid preparations such as granules, powders and the like. Because of ease of use and high compliance, tablets and capsules provide an advantageous oral dosage form for many medical conditions.

  Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs, etc. Suitable carriers and additives include water, alcohols, fats, glycols, preservatives, flavors Agents, coloring agents, suspending agents and the like. Typical preparations for parenteral administration contain the active ingredient together with sterile water or a parenterally acceptable oil, such as polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil, and are soluble or preserved Other additives that contribute to can also be included. In the case of a solution, it can be lyophilized to a powder and then reconstituted immediately before use. In the case of dispersions and suspensions, suitable carriers and additives include aqueous gums, cellulose, silicates and fats.

  Pharmaceutical compositions according to embodiments of the present invention can be oral, rectal, topical, nasal, inhalation (eg, via aerosol), oral (eg, sublingual), intravaginal, topical (ie, both skin and mucosal surfaces, such as Suitable for airway surface), transdermal administration and parenteral (eg, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intrathecal, intracerebral, intracranial, intraarterial or intravenous) The most appropriate route in a given case will vary depending on the nature and severity of the condition to be treated and on the nature of the particular active agent used. The pharmaceutical compositions of the invention are particularly suitable for oral, sublingual, parenteral, implant, nasal and inhalation administration.

Injectable compositions may be suitable carriers such as propylene glycol-water, isotonic water, sterile water for injection (USP), emulPhor ^™ -alcohol water, cremophor-EL ^™ or other suitable known in the art. Active ingredients together with a carrier. These carriers may be used alone or in combination with other conventional solubilizing goods such as ethanol, glycol or other materials known in the art.

  When the compounds of the invention are applied in the form of a solution or injection, the compounds can be used by dissolving or suspending in any conventional diluent. Diluents can include, for example, physiological saline, Ringer's solution, glucose aqueous solution, dextrose aqueous solution, alcohol, fatty acid ester, glycerol, glycol, fats and oils derived from plant or animal raw materials, paraffin and the like. These formulations can be prepared by any conventional method known in the art.

  Compositions for nasal administration can be formulated as aerosols, drops, powders and gels. Aerosol formulations typically include a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent. Such formulations are typically provided in single or multi-dose quantities in a sterilized form during the sealing phase. The sealed container can be a cartridge or refill for use with an atomizer. Alternatively, the sealed container may be a uniform dispensing device, such as a single use nasal inhaler, a pump atomizing device or an aerosol dispenser with a metering valve set to deliver a therapeutically effective amount, This is intended to be evacuated after the contents are fully used. If the dosage form comprises an aerosol dispenser, it will contain a high pressure gas, such as compressed gas, air, etc., or an organic high pressure gas, such as fluorochlorohydrocarbon or fluorohydrocarbon.

  Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastels, where the active ingredient is formulated with a carrier such as a sugar and acacia, tragacanth or gelatin and glycerin.

  Compositions for rectal administration include suppositories containing a conventional suppository base such as cocoa butter.

  Compositions suitable for transdermal administration include ointments, gels and patches.

  Other compositions known in the art, such as plasters, may also be applied for transdermal or subcutaneous administration.

  Furthermore, when preparing such pharmaceutical compositions comprising the active ingredient in a mixture with the ingredients necessary for formulation of the composition, other conventional pharmaceutically acceptable additives such as Excipients, stabilizers, preservatives, wettable powders, emulsifiers, lubricants, sweeteners, colorants, flavoring agents, tonicity-imparting agents, buffering agents, antioxidants and the like can also be incorporated. Additives include starch, sucrose, fructose, dextrose, lactose, glucose, mannitol, sorbitol, precipitated calcium carbonate, crystalline cellulose, carboxymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, Examples thereof include sodium metabisulfite.

  In another embodiment, the present invention provides a kit comprising one or more containers comprising a pharmaceutical dosage unit comprising an effective amount of one or more compounds of the present invention.

  If an aqueous suspension or elixir is desired for oral administration, the essential active ingredients contained therein are various sweetening or flavoring agents, coloring agents or dyes, and optionally emulsifying or suspending agents, and It can be combined with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof.

  The compositions described herein can be administered as part of a sustained release formulation. Such formulations may be manufactured using generally well-known techniques and, for example, by oral, rectal or transdermal delivery systems, or implant treatment of the formulation or therapeutic device at one or more desired target sites Can be administered. The sustained release formulation contains a therapeutic composition comprising the therapeutic agent of the present invention alone or a second therapeutic agent dispersed in a carrier matrix or contained within a reservoir surrounded by a rate controlling membrane. . Carriers for use in such formulations are biocompatible and can be biodegradable. According to one embodiment, the sustained release formulation may result in a relatively constant level of active composition release. According to another embodiment, the sustained release formulation can be contained within a device that can be driven by a subject or medical personnel at the onset of a particular condition, for example, to deliver a predetermined dose of the therapeutic composition. . The amount of therapeutic composition contained within the sustained release formulation will depend on the site of the implant treatment, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

In certain embodiments, the compositions of the invention for the treatment of neuropathic pain and neuropsychiatric disorders are administered using formulations and routes of administration that facilitate delivery of the administered composition to the central nervous system. Administration compositions such as NKCC1 antagonists may be formulated to facilitate crossing the blood brain barrier as described above, or may be co-administered with agents that cross the blood brain barrier. The administration compositions may be delivered in a liposome formulation to pass through the example blood-brain barrier, or other compounds that cross the blood brain barrier, for example bradykinin, bradykinin analogs or derivatives, or other compounds such SERAPORT ^TM coadministered Can do. Alternatively, the administration composition of the present invention may be delivered using a spinal tap that places the administration composition directly into the circulating cerebrospinal fluid. For some therapeutic conditions, transient or permanent disruption of the blood brain barrier may occur, and a special formulation of the administered composition to cross the blood brain barrier may not be required. The inventors have shown, for example, that an instantaneous intravenous injection of furosemide 20 mg reduces or eliminates both spontaneous seizure activity and electrical stimulation-induced sputum-like activity in human patients refractory to antidepressants (AEDs). (Haglund & Hochman J. Neurophysiol. (Feb. 23, 2005) doi: 10.1152 / jn.00944.2004).

  The route and frequency and dosage of administration of the therapeutic compositions disclosed herein will vary from indication to indication and from individual to individual, and from commonly available information and patient monitoring and standard techniques Can be easily determined by a physician by adjusting the dose and usage as appropriate. In general, a suitable dose and usage is one that provides a sufficient amount of the active composition to provide therapeutic and / or prophylactic benefits. Dosage and usage can be established by monitoring improved clinical outcome in administered patients as compared to non-administered patients.

  The term “effective amount” or “effective” is intended to refer to a dose that results in alleviation of symptoms of a disease or disorder as sensed through clinical trials and evaluation, patient observation, and the like. “Effective amount” or “effective” can further refer to a dose that induces a detectable change in biological or chemical activity. Those skilled in the art can detect the detectable change and / or have further quantified the relevant mechanism or process. Furthermore, an “effective amount” or “effective” refers to an amount that maintains a desired physiological state, ie, reduces or prevents significant deterioration and / or promotes improvement of the target state. it can. The therapeutically effective dose and usage will depend on the condition of the patient to be treated, the severity of the condition, and the general condition. Because the pharmacokinetic and pharmacodynamic characteristics of the administration composition of the present invention vary in different patients, the preferred method for determining the therapeutically effective amount in patients is to gradually increase the dose and monitor clinical laboratory indicators That is. In the case of combination therapy, two or more drugs are co-administered such that each of the drugs is present in a therapeutically effective amount for a time sufficient to provide a therapeutic or prophylactic effect. The term “simultaneous administration” is intended to encompass the simultaneous or sequential administration of two or more agents in the same formulation or unit dosage form or in separate formulations. Appropriate dosages and usage for the treatment or prevention of acute episodes, chronic conditions will necessarily vary to suit the patient's condition.

  By way of example, for the treatment of neuropathic pain, furosemide may be administered orally to a patient in an amount of 10-40 mg, preferably 3 times a day, 40 mg at a frequency of 1-3 times per day. . In another example, bumetanide may be administered orally for the treatment of neuropathic pain in an amount of 1-10 mg at a frequency of 1-3 times per day. Those skilled in the art will know that smaller doses may be used, for example, in pediatric applications.

  In another embodiment, the bumetanide analogue according to the invention may be administered in an amount of 1.5-6 mg daily, for example 1 tablet or 1 capsule 3 times a day. In some embodiments, a furosemide analog according to the invention may be administered in an amount of 60-240 mg / day, for example, 1 tablet or 1 capsule 3 times per day. In another embodiment, the piretanide analog according to the invention may be administered in an amount of 10-20 mg daily, for example 1 tablet or 1 capsule once daily. In some embodiments, the azosemide analog according to the invention may be administered in an amount of 60 mg per day. In another embodiment, the torsemide analog of the present invention may be administered in an amount of 10-20 mg daily, for example, 1 tablet or 1 capsule once daily. Of course, especially for IV administration, lower doses may be administered.

  The methods and systems of the invention can also be used to evaluate candidate compounds and rewards for the treatment and / or prevention of neuropathic pain and neuropsychiatric disorders. Various techniques can be used to generate candidate compounds potentially having the desired NKCC1 cotransporter antagonist activity. Candidate compounds can be made using procedures well known in the art of synthetic organic chemistry. Structure-activity relationships and molecular modeling techniques are useful for the purpose of modifying known NKCC1 antagonists such as furosemide, bumetanide, ethacrynic acid and related compounds to confer the desired activity and specificity. Methods for screening candidate compounds for a desired activity are described in US Pat. Nos. 5,902,732, 5,976,825, 6,096,510 and 6,319,682, which are incorporated herein by reference in their entirety. Has been.

  Candidate compounds can be screened for NKCC1 antagonist activity using various types of cultured cells such as glial cells, neuronal cells, kidney cells, etc., or in an in situ animal model, using the screening response of the present invention. Screening approaches to discover chloride cotransporter antagonist activity can, for example, increase the ion balance of tissue culture samples or in situ extracellular space in animal models by generating anionic chloride concentrations that are higher than “normal”. Modify. A candidate agent is administered by measuring the geometric and / or optical properties of a sample of cells or tissue subjected to this modified ion balance. After administration of the candidate agent, whether ionic imbalances remain by monitoring the corresponding geometric and / or optical properties of the cell or tissue sample, or in the extracellular and intracellular spaces Determine whether the cells responded by altering the ion balance. If an ionic imbalance remains, the candidate agent may be a chloride cotransporter antagonist. Screening using various types of cells or tissues has high levels of glial chloride cotransporter antagonist activity and low levels of neuronal and renal cell chloride cotransporter antagonist activity Candidate compounds are discovered. Similarly, effects on different types of cell and tissue systems can be evaluated.

  In addition, the candidate compound may stimulate or induce a condition such as neuropathic pain in situ in an animal model, and the geometric and / or optical properties of the cell or tissue sample during the stimulation of the condition. Monitoring, administering the candidate agent, then monitoring the geometric and / or optical properties of the cell or tissue after administration of the candidate agent, and the geometric and / or sample of the cell or tissue sample It can be evaluated by comparing the optical properties and examining the action of the candidate compound. A test of the efficacy of the administered composition for alleviating neuropathic pain is described in Bennett, Hosp. Pract. (Off Ed). 33: 95-98, 1998 can be used using well known methods.

  As described above, the composition used in the methods of the present invention comprises an antibody that specifically binds to NKCC1, or an antigen-binding fragment thereof; a soluble ligand that binds to NKCC1, an antisense oligonucleotide of NKCC1, and a short that is specific to NKCC1. A therapeutic agent selected from the group consisting of strand-interfering RNA molecules (siRNA or RNAi) may be included.

  Antibodies that specifically bind to NKCC1 are known in the art and are described in Alpha Diagnostic International, Inc. (San Antonio, Tex. 78238). The “antigen-binding site” or “antigen-binding fragment” of an antibody refers to the portion of the antibody that participates in antigen binding. The antigen binding site is formed by amino acid residues in the N-terminal variable (V) region of the heavy (H) chain and light (L) chain. Three highly divergent stretches in the V and V regions of the heavy and light chains are termed “hypervariable regions”, which are between the more conserved flanking stretches known as “framework regions” or “FRs”. Has been inserted. That is, the term “FR” refers to the naturally occurring amino acid sequence adjacent to and between the hypervariable regions of an immunoglobulin. In the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are each relatively arranged in a three-dimensional space to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity determining regions” or “CDRs”.

Many molecules are known in the art that contain an antigen binding site capable of exhibiting the binding properties of an antibody molecule. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to form several fragments, two of which (“F (ab)” fragments) each contain a covalent heterozygote containing an intact antigen binding site. Includes dimers. The enzyme pepsin can cleave IgG molecules to form several fragments, the contained “F (ab ′) ₂ ” fragment containing both antigen binding sites. “Fv” fragments can be formed by preferential proteolytic cleavage of IgM, IgG, or IgA immunoglobulin molecules, but are more generally derived using recombinant techniques known in the art. Fv fragments are non-covalent V _H :: V _L heterodimers that contain an antigen binding site that retains most of the antigen recognition and binding ability of the native antibody molecule (Inbar et al. Proc. Natl. Acad). Sci. USA 69: 2659-2662, 1972; Hochman et al. Biochem 15: 2706-2710, 1976; and Ehrlich et al.

  Humanized antibodies that specifically bind to NKCC1 can also be used in the methods of the invention. Many humanized antibody molecules containing an antigen binding site derived from non-human immunoglobulin have been reported, for example, chimeric antibodies carrying a rodent V region and its associated CDR fused to a human constant domain ( Winter et al. Nature 349: 293-299, 1991; Lobglio et al. Proc. Natl. Acad. Sci. USA 86: 4220-4224, 1989; Shaw et al. J Immunol.138: 4534-4538, 1987; Brown et al. Cancer Res. 47: 3577-3583, 1987); rodent CDRs (Riechmann et al. Na) grafted into a human support framework prior to fusion with the appropriate human antibody constant domain in. true 332: 323-327, 1988; Verhoeyen et al. Science 239: 1534-1536, 1988; and Jones et al. Nature 321: 522-525, 1986); and supported by recombinantly modified rodent FRs. Rodent CDRs (European Patent Publication 519,596 published 23 December 1992) are included. These “humanized” molecules minimize the undesirable immunological response to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic application of these parts in human recipients. Designed.

  Modulating the activity of NKCC1 may alternatively be achieved by reducing or repressing the expression of the polypeptide, which is achieved by interfering with the transcription and / or translation of the corresponding polynucleotide. Can do. Polypeptide expression is for example by introducing antisense expression vectors, antisense oligodeoxyribonucleotides, antisense phosphorothioate oligodeoxyribonucleotides, antisense oligonucleotides or antisense phosphorothioate oligoribonucleotides; or are well known in the art It can be suppressed by other means. All such antisense polynucleotides are referred to herein as “antisense oligonucleotides”.

  Antisense oligonucleotides for use in the methods of the invention have sufficient complementarity to the NKCC1 polynucleotide to specifically bind to the polynucleotide. In order for an antisense oligonucleotide to be effective in the methods of the invention, the sequence of the antisense oligonucleotide need not be 100% complementary to the polynucleotide. Rather, when antisense oligonucleotide binding to the polynucleotide interferes with the normal function of the polynucleotide and eliminates its use, and nonspecific binding of the oligonucleotide to other non-target sequences is avoided. In addition, antisense oligonucleotides are sufficiently complementary. The design of suitable antisense oligonucleotides is well known in the art. Oligonucleotides that are complementary to the 5 'end of the message, such as the 5' untranslated sequence up to and including the AUG start codon, should function most efficiently in translational repression. However, oligonucleotides that are complementary to either the 5 'or 3' untranslated uncoding region of the targeted polynucleotide may also be used. Cell permeability and activity of antisense oligonucleotides can be determined by appropriate chemical modifications, such as phenoxazine substituted C-5 propynyl uracil oligonucleotides (Flanagan et al., Nat. Biotechnol. 17: 48-52, 1999) or 2′- It can be enhanced by the use of O- (2-methoxy) ethyl (2′-MOE) -oligonucleotide (Zhang et al., Nat. Biotechnol. 18: 862-867, 2000). The use of techniques using antisense oligonucleotides is well known in the art, see, eg, Robinson-Benion et al. (Methods in Enzymol. 254: 363-375, 1995) and Kawasaki et al. (Artific. Organs 20: 836-848, 1996).

  The expression of NKCC1 polypeptide can be suppressed more specifically than by methods such as RNA interference (RNAi). A discussion of this approach is described in Science, 288: 1370-1372, 2000. In other words, traditional methods of gene suppression using antisense RNA or DNA block the synthesis of the corresponding protein by binding to the reverse sequence of the gene of interest so that binding interferes with subsequent cellular processes. It works by RNAi also acts on post-translational levels and is sequence specific, but represses gene expression much more efficiently. Exemplary methods for controlling or modifying gene expression are described in WO99 / 49029, WO99 / 53050 and WO01 / 75164, which are incorporated herein by reference. In these methods, post-translational gene silencing is performed by a sequence-specific RNA degradation process that results in rapid degradation of transcripts of sequence-related genes. Studies have shown that double-stranded RNA acts as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14: 255-258, 1998). Gene constructs that produce transcripts with self-complementary regions are particularly efficient in gene silencing.

  It has been shown that one or more ribonucleases specifically bind to double stranded RNA and cleave into short fragments. The ribonucleases remain associated with these fragments, which in turn bind specifically to complementary mRNA, i.e. specifically to the transcribed mRNA strand for the gene of interest. The mRNA for a gene is also degraded by ribonuclease into short fragments that prevent gene translation and expression. Furthermore, mRNA polymerase has the effect of promoting the synthesis of many copies of short fragments, which exponentially increases the efficiency of the system. A unique feature of RNAi is that silencing is not limited to the cell in which it is initiated. Gene silencing may be disseminated to other parts of the organism.

  That is, NKCC1 polynucleotides are used to form gene silencing constructs and / or gene specific self-complementary double stranded RNA sequences that can be used in the methods of the invention using delivery methods known in the art. Can be used. The gene construct can be used to express a self-complementary RNA sequence. Alternatively, the cell can be contacted with a gene-specific double stranded RNA molecule such that the RNA molecule is internalized within the cell protoplast and exhibits a gene silencing effect. The double stranded RNA must have sufficient homology to the NKCC1 gene to mediate RNAi without affecting the expression of non-target genes. Double-stranded DNA is at least 20 nucleotides long, preferably 21-23 nucleotides long. Preferably, the double-stranded RNA specifically corresponds to the polynucleotide of the present invention. The use of short interfering RNA (siRNA) molecules 21 to 23 nucleotides long to suppress gene expression in mammalian cells is described in WO01 / 75164. Means for designing optimal inhibitory siRNA are described in DNAengine Inc. Includes those available from (Seattle, WA.).

  One RNAi approach uses a gene construct in which the sense and antisense sequences are located in a region flanked by appropriately spliced intron sequences with donor and acceptor splicing sites. Alternatively, spacer sequences of various lengths can be used to isolate self-complementary regions of sequences within the construct. During processing of the gene construct transcript, intron sequences are spliced out, allowing sense and antisense sequences and splice junction sequences to bind, forming double-stranded RNA. A ribonuclease is then selected that binds to and cleaves the double stranded RNA, thereby initiating a cascade of events that results in the degradation of specific mRNA gene sequences, splicing specific genes.

  For in vivo use, gene constructs, antisense oligonucleotides or RNA molecules can be administered by a variety of procedures known in the art (eg, Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143- 198, 1998 and references cited therein). Viral and non-viral delivery methods are used for gene therapy. Useful viral vectors include, for example, adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia virus and tripox virus. For example, by coupling adenovirus to the DNA polylysine complex and by utilizing receptor-mediated endocytosis for selective targeting, the efficiency of gene targeting to tumor cells using adenovirus vectors is increased. (See, for example, Curiel et al., Hum. Gene Ther., 3: 147-154, 1992; and Cristiano & Curiel, Cancer Gene Ther. 3: 49-57, 1996). Non-viral methods for delivering polynucleotides are described in Chang & Seymour, (Eds) Curr. Opin. Mol. Ther. , Vol. 2, 2000. These methods include contacting cells with naked DNA, polynucleotide polyplexes with cationic liposomes or cationic polymers, and dendrimers for systemic administration (Chang & Seymour, supra). Liposomes can be modified by incorporation of ligands that recognize cell surface receptors and allow targeting of specific receptors for uptake by receptor-mediated endocytosis (eg, Xu et al., Mol. Genet). Metab., 64: 193-197; 1998; and Xu et al., Hum. Gene Ther., 10: 2941-22952;

  Tumor targeting bacteria such as Salmonella are potentially useful for the delivery of genes to tumors after systemic administration (Low et al., Nat. Biotechnol. 17: 37-41, 1999). Bacteria can be engineered ex vivo, for example, to penetrate into mammalian epithelial cells in vivo and deliver DNA with high efficiency (eg, Grillot-Courvalin et al., Nat. Biotechnol. 16: 862). 866, 1998). Degraded and stabilized oligonucleotides can be encapsulated in liposomes and delivered to a patient by injection intravenously or directly into a target site (eg, the source of neuropathic pain). Alternatively, retroviral or adenoviral vectors or naked DNA that expresses antisense RNA for the polypeptides of the invention can be administered to the patient. Suitable techniques for using such methods are well known in the art.

  The administration compositions and methods of the present invention have been described above with respect to certain preferred embodiments. The following examples illustrate the results of specific experiments and are not intended to limit the invention in any way.

Example 1
Methyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumethanide methyl ester)
To a slurry of bumetanide (1.2 g, 3.29 mmol) in methanol (12 mL) under nitrogen was added a mixture of thionyl chloride (70 uL) in methanol (6 mL) over 5 minutes. After stirring for 5 minutes, the reaction mixture became dissolved. The reaction mixture was stirred for an additional 30 minutes, at which point the reaction was complete as confirmed by thin layer chromatography (TLC). Methanol was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate, water and brine. Ethyl acetate was dried over anhydrous magnesium sulfate and concentrated to give 1.1 g (89%) of methyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate as a white solid.

Example 2
Cyanomethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (Bumetanide cyanomethyl ester)
Bumetanide (1.0 g, 2.7 mmol) was dissolved in dimethylformamide (DMF) and chloroacetonitrile (195 uL, 2.7 mmol) was added followed by triethylamine (465 uL). The reaction mixture was heated to 100 ° C. for 12 hours, at which time TLC and liquid chromatography coupled mass spectrometer (LC / MC) indicated that the reaction was complete. The reaction mixture was cooled to room temperature, dissolved in dichloromethane, washed with water, saturated ammonium chloride and reduced to a slurry. Water (25 mL) was added to the slurry and the crude product was precipitated as an off-white solid. Pure cyanomethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (850 mg) was obtained via recrystallization in acetonitrile.

Example 3
Benzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide benzyl ester)
Bumetanide (1.15 g, 3.15 mmol) was dissolved in dimethylformamide (DMF, 10 mL) and benzyl chloride (400 uL, 2.8 mmol) was added followed by triethylamine (480 uL). The reaction mixture was heated to 80 ° C. for 12 hours, at which time TLC and LC / MS indicated that the reaction was complete. The reaction mixture was cooled to room temperature, dissolved in dichloromethane, washed with water, saturated ammonium chloride, and concentrated to a thick slurry. Water (25 mL) was added to the slurry. The resulting solid was filtered and dried in a vacuum oven at 50 ° C. for 12 hours to give 1.0 g (80%) of benzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

Example 4
2- (4-morpholino) ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide morpholinoethyl ester)
Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 12 mL), 4- (2-chloroethyl) morpholine hydrochloride (675 mg, 3.62 mmol), then triethylamine (1 mL) and sodium iodide. (500 mg, 3.33 mmol) was added. The reaction mixture was heated to 95 ° C. for 8 hours, at which time TLC and LC / MS indicated that the reaction was complete. The reaction mixture was cooled to room temperature, dissolved in dichloromethane, washed with water, saturated ammonium chloride and concentrated to dryness. After purification via Biotage flash chromatography, purification elution and evaporation under vacuum, 2- (4-morpholino) ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate was obtained as a white solid ( 600 mg, 62%).

Example 5
3- (N, N-dimethylaminopropyl) 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate [Bumetanide 3- (dimethylaminopropyl) ester]
In the same manner as in Example 31, bumetanide was reacted with 3- (dimethylamino) propyl hydrochloride, triethylamine and sodium iodide in dimethylformamide (DMF) to give 3- (N, N-dimethylaminopropyl) 3- Aminosulfonyl-5-butylamino-4-phenoxybenzoate can be obtained.

Example 6
N, N-diethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide N, N-diethylglycolamide ester)
Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (12 mL), 2-chloro-N, N-diethylacetamide (500 mg, 3.35 mmol), then triethylamine (0.68 mL) and iodine. Sodium chloride (500 mg, 3.33 mmol) was added. The reaction mixture was heated to 95 ° C. for 8 hours, at which time TLC and LC / MS indicated that the reaction was complete. The reaction mixture was cooled to room temperature, dissolved in dichloromethane, washed with water, saturated ammonium chloride, and reduced to a thick slurry. Water (25 mL) was added to the slurry and the resulting solid was precipitated from the solution. The product was filtered and dried in a vacuum oven at 50 ° C. for 12 hours to give 1.0 g of N, N-diethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

Example 7
N, N-diethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide diethylamide)
Bumetanide (1.16 g, 3.2 mmol) was dissolved in dichloromethane (10 mL) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 690 mg, 3.6 mmol) was added. After 5 minutes, N-hydroxybenzotriazole (HOBt, 498 mg, 3.6 mmol) was added and the solution was stirred for an additional 5 minutes. Diethylamino (332 uL, 3.2 mmol) was added and the reaction mixture was stirred for 2 hours. The reaction mixture was washed with saturated sodium bicarbonate, water and brine and dried over magnesium sulfate. Dichloromethane was removed under reduced pressure to give 860 mg (65%) of pure N, N-diethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

Example 8
N, N-dibenzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide dibenzylamide)
Bumetanide (960 mg, 2.6 mmol) was dissolved in dimethylformamide (DMF, 10 mL) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 560 mg, 3.6 mmol) was added. After 10 minutes, 1-hydroxybenzotriazole (HOBt, 392 mg, 2.9 mmol) was added and the solution was stirred for an additional 10 minutes. Dibenzylamine (1 mL, 5.2 mmol) was added and the reaction mixture was stirred for 2 h, at which point the reaction was complete by LC / MS. The reaction mixture was poured into saturated ammonium chloride (20 mL) and extracted with ethyl acetate (2 × 100 mL). Ethyl acetate was washed with saturated sodium bicarbonate, water and brine and dried over anhydrous sodium sulfate. Ethyl acetate was removed under reduced pressure to give 1.0 g (75%) of N, N-dibenzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate as a white solid.

Example 9
Benzyltrimethylammonium 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide benzyltrimethylammonium salt)
To a solution of benzyltrimethylammonium hydroxide (451 mg, 2.7 mmol) in water (10 mL) was added bumetanide (1 g, 2.7 mmol) over 5 minutes. After stirring for 10 minutes, the reaction mixture became clear. Water was removed under reduced pressure to give a crude colorless oil. The pure product was obtained from recrystallization of the oil using water and heptane to give 690 mg of benzyltrimethylammonium 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate as light pink crystals.

Example 10
Cetyltrimethylammonium 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (Bumetanide cetyltrimethylammonium salt)
In the same manner as in Example 9, bumetanide can be reacted with cetyltrimethylammonium hydroxide in water to give cetyltrimethylammonium 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

Example 11
N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (Bumetanide N, N-dimethylglycolamide ester)
Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 10 mL), 2-chloro-N, N-dimethylamide (410 uL, 3.9 mmol), then triethylamine (0.70 mL) and Sodium iodide (545 mg, 3.6 mmol) was added. The reaction mixture was heated to 50 ° C. for 10 hours, at which time TLC and LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed with saturated sodium bicarbonate, water and brine, and dried over anhydrous magnesium sulfate. Ethyl acetate was removed under reduced pressure and the product was purified by flash chromatography to yield 685 mg (60%) of pure N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate. It was.

Example 12
t-Butylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide pibaxetyl ester)
Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 10 mL) and chloromethyl pivalate (575 uL, 3.9 mmol) followed by triethylamine (0.70 mL) and sodium iodide (545 mg). 3.6 mmol) was added. The reaction mixture was heated to 50 ° C. for 10 hours, at which time TLC and LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed with saturated sodium bicarbonate, water and brine, and dried over anhydrous sodium sulfate. Ethyl acetate was removed under reduced pressure and the product was purified by flash chromatography to give 653 mg (60%) of pure t-butylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

Example 13
Ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (Bumetanide propaxetyl ester)
In a manner similar to Example 12, bumetanide is reacted with chloromethylpropionate, trimethylamine and sodium iodide in dimethylformamide (DMF) to give ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxy. Benzoate can be obtained.

Example 14
Methyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide methyl ester)
Piretanide can be reacted with thionyl chloride and methanol in the same manner as in Example 1 to give methyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate.

Example 15
Cyanomethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide cyanomethyl ester)
In the same manner as in Example 2, piretanide can be reacted with chloroacetonitrile in DMF to give cyanomethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate.

Example 16
Benzyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide benzyl ester)
In the same manner as in Example 3, piretanide can be reacted with benzyl chloride in DMF to give benzyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate.

Example 17
2- (4-morpholino) ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide morpholinoethyl ester)
In a manner similar to Example 4, piretanide was reacted with 4- (2-chloroethyl) morpholino hydrochloride, triethylamine and sodium iodide in DMF to give 2- (4-morpholino) ethyl 3-aminosulfonyl-4-phenoxy- 5- (1-Pyrrolidinyl) benzoate can be obtained.

Example 18
3- (N, N-dimethylaminopropyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate [pyretanide 3- (dimethylaminopropyl) ester]
In a similar manner to Example 31, piretanide was reacted with 3- (dimethylamino) propyl chloride hydrochloride, triethylamine and sodium iodide in dimethylformamide (DMF) to give 3- (N, N-dimethylaminopropyl 3-amino Sulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate can be obtained.

Example 19
N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide N, N-diethylglycolamide ester)
In a manner similar to Example 6, piretanide was reacted with 2-chloro-N, N-diethylacetamide, triethylamine and sodium iodide in dimethylformamide (DMF) to give N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl. -4-Phenoxy-5- (1-pyrrolidinyl) benzoate can be obtained.

Example 20
N, N-diethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide diethylamide)
In a manner similar to Example 7, piretanide can be reacted with EDC, HOBt and diethylamine in DMF to give N, N-diethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate. .

Example 21
N, N-dibenzyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide dibenzylamide)
Reacting Piretanide with EDC, HOBt and dibenzylamine in DMF in a manner similar to Example 8 to obtain N, N-dibenzyl-3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate Can do.

Example 22
Benzyltrimethylammonium 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide benzyltrimethylammonium salt)
Piretanide can be reacted with benzyltrimethylammonium in the same manner as in Example 9 to obtain benzyltrimethylammonium 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate.

Example 23
Cetyltrimethylammonium 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide cetyltrimethylammonium salt)
Piretanide can be reacted with cetyltrimethylammonium in the same manner as in Example 10 to obtain cetyltrimethylammonium 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate.

Example 24
N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide N, N-dimethylglycolamide ester)
In a manner similar to Example 11, piretanide was reacted with 2-chloro-N, N-dimethylacetamide, triethylamine and sodium iodide in DMF to give N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy. -5- (1-Pyrrolidinyl) benzoate can be obtained.

Example 25
t-Butylcarbonyloxymethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide pibaxetyl ester)
In a manner similar to Example 12, piretanide was reacted with chloromethyl pivalate, triethylamine and sodium iodide in DMF to give t-butylcarbonyloxymethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl). Benzoate can be obtained.

Example 26
Ethylcarbonyloxymethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (pyretanide propaxetyl ester)
In the same manner as in Example 13, pyrethanide was reacted with chloromethylpropionate, triethylamine and sodium iodide in DMF to give ethylcarbonyloxymethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate. Can be obtained.

Example 27
Ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide ethyl ester)
Bundgaard, H.M. , Norgaad, T .; And Nielsen, N .; M.M. , Int. J. et al. Pharmaceutics, 1988, 42, 217-224 can be used to prepare ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. Melting point: 163-165 ° C.

Example 28
Cyanomethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide cyanomethyl ester)
In the same manner as in Example 2, furosemide can be reacted with chloroacetonitrile in DMF to give cyanomethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate.

Example 29
Benzyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide benzyl ester)
In the same manner as in Example 3, furosemide can be reacted with benzyl chloride in DMF to give benzyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate.

Example 30
2- (4-morpholino) ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide morpholinoethyl ester)
Mork, N .; , Bundgaard, H .; Shalmi, M .; And Christensen, S .; , Int. J. et al. Pharmaceutics, 1990, 60, 163-169 can be used to prepare ethyl 2- (4-morpholino) ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. Mp 134-135 ° C.

Example 31
3- (N, N-dimethylaminopropyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide 3- (dimethylaminopropyl) ester)
Mork, N .; , Bundgaard, H .; Shalmi, M .; And Christensen, S .; , Int. J. et al. The method of Pharmaceuticals, 1990, 60, 163-169 can be used for the preparation of 3- (N, N-dimethylaminopropyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate, mp 212. ~ 213 ° C.

Example 32
N, N-diethylaminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide N, N-diethylglycolamide ester)
Mork, N .; , Bundgaard, H .; Shalmi, M .; And Christensen, S .; , Int. J. et al. Pharmaceutics, 1990, 60, 163-169 can be used to prepare N, N-diethylaminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. Mp 135-136 ° C.

Example 33
N, N-diethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide diethylamide)
In the same manner as in Example 7, furosemide is reacted with EDC, HOBt and diethylamine in DMF to give N, N-diethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. be able to.

Example 34
N, N-dibenzyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide dibenzylamide)
In the same manner as in Example 8, furosemide was reacted with EDC, HOBt and dibenzylamine in DMF to give N, N-dibenzyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. Can be obtained.

Example 35
Benzyltrimethylammonium 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide benzyltrimethylammonium salt)
In the same manner as in Example 9, furosemide can be reacted with benzyltrimethylammonium hydroxide to give benzyltrimethylammonium 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate.

Example 36
Cetyltrimethylammonium 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemidocetyltrimethylammonium salt)
In the same manner as in Example 10, furosemide can be reacted with cetyltrimethylammonium hydroxide to obtain cetyltrimethylammonium 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate.

Example 37
N, N-dimethylaminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide N, N-dimethylglycolamide ester)
Bundgaard, H.M. , Norgaad, T .; And Nielsen, N .; M.M. , Int. J. et al. Pharmaceutics, 1988, 42, 217-224 can be used for the preparation of N, N-dimethylaminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. 193-194 ° C.

Example 38
t-Butylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide pibaxetyl ester)
Mork, N .; , Bundgaard, H .; Shalmi, M .; And Christensen, S .; , Int. J. et al. The method of Pharmaceuticals, 1990, 60, 163-169 can be used for the preparation of t-butylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate.

Example 39
Ethylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide propaxyl ester)
Mork, N .; , Bundgaard, H .; Shalmi, M .; And Christensen, S .; , Int. J. et al. The method of Pharmaceuticals, 1990, 60, 163-169 can be used for the preparation of ethylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate. Mp 141-142 ° C.

Example 40
5- [1- (t-Butylcarbonyloxymethyl) -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
In a manner similar to Example 12, azosemide is reacted with chloromethyl pivalate, triethylamine and sodium iodide in DMF to give 5- [1- (t-butylcarbonyloxymethyl) -1H-tetrazol-5-yl]. -2-Chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide can be obtained.

Example 41
2-Chloro-5- [1- (ethylcarbonyloxymethyl) -1H-tetrazol-5-yl] -4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
In a manner similar to Example 12, azosemide was reacted with chloromethylpropionate, triethylamine and sodium iodide in DMF to give 2-chloro-5- [1- (ethylcarbonyloxymethyl) -1H-tetrazole-5. -Il] -4-[(2-thienylmethyl) amino] benzenesulfonamide can be obtained.

Example 42
2-Chloro-5- [1- (hydroxymethyl) -1H-tetrazol-5-yl] -4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
Azosemide is reacted with methylene chloride, methylene chloride-DMF mixture or formaldehyde in DMF to give 2-chloro-5- [1- (hydroxymethyl) -1H-tetrazol-5-yl] -4-[(2-thienylmethyl) Amino] benzenesulfonamide can be obtained.

Example 43
2-Chloro-5- [1- (methoxymethyl) -1H-tetrazol-5-yl] -4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
Azosemide is reacted with formaldehyde and methanol in methylene chloride, methylene chloride-DMF mixture or DMF to give 2-chloro-5- [1- (hydroxymethyl) -1H-tetrazol-5-yl] -4-[(2- Thienylmethyl) amino] benzenesulfonamide can be obtained.

Example 44
2-Chloro-5- [1- (methylthiomethyl) -1H-tetrazol-5-yl] -4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
Azosemide is reacted with formaldehyde and methanethiol in methylene chloride, methylene chloride-DMF mixture or DMF to give 2-chloro-5- [1- (methylthiomethyl) -1H-tetrazol-5-yl] -4-[(2 -Thienylmethyl) amino] benzenesulfonamide can be obtained.

Example 45
5- [1- (Benzyloxymethyl) -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide)
Azosemide is reacted with benzyl chloromethyl ether, triethylamine and sodium iodide in DMF to give 5- [1- (benzyloxymethyl) -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienyl). Methyl) amino] benzenesulfonamide can be obtained.

Example 46
Benzyltrimethylammonium salt of 2-chloro-5- (1H-tetrazol-5-yl) -4-[(2-thienylmethyl) amino] benzenesulfonamide (azosemide benzyltrimethylammonium salt)
In the same manner as in Example 9, azosemide is reacted with benzyltrimethylammonium hydroxide in water to give 2-chloro-5- (1H-tetrazol-5-yl) -4-[(2-thienylmethyl) amino]. Benzyltrimethylammonium salt of benzenesulfonamide can be obtained.

Example 47
Cetyltrimethylammonium salt of 2-chloro-5- (1H-tetrazol-5-yl) -4-[(2-thienylmethyl) amino] benzenesulfonamide (azosemidocetyltrimethylammonium salt)
In the same manner as in Example 9, azosemide is reacted with cetyltrimethylammonium hydroxide in water to give 2-chloro-5- (1H-tetrazol-5-yl) -4-[(2-thienylmethyl) amino]. A cetyltrimethylammonium salt of benzenesulfonamide can be obtained.

Example 48
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium t-butylcarbonyloxymethochloride (pyridinium-substituted torsemide salt)
In a similar manner to Example 12, torsemide was reacted with chloromethyl pivalate, triethylamine and sodium iodide in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium t-butyl. Carbonyloxymethochloride and some 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium t-butylcarbonyloxy-methiodide can be obtained.

Example 49
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium ethylcarbonyloxymethochloride (pyridinium-substituted torsemide salt)
In a manner similar to Example 12, torsemide was reacted with chloromethylpropinate, triethylamine and sodium iodide in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium ethylcarbonyloxy. Metochloride and some 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium ethylcarbonyloxy-methiodide can be obtained.

Example 50
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium benzyloxymethochloride (pyridinium-substituted torsemide salt)
In the same manner as in Example 3, torsemide is reacted with benzyl chloromethyl ether and triethylamine in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium benzyloxymethochloride. Can do.

Example 51
3-Isopropylcarbamylsulfonamide-4- (3′-methylphenyl) aminopyridinium methoxymethochloride (pyridinium-substituted torsemide salt)
In the same manner as in Example 3, torsemide can be reacted with methyl chloromethyl ether and triethylamine in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium methoxymethochloride. it can.

Example 52
3-Isopropylcarbamylsulfonamide-4- (3′-methylphenyl) aminopyridinium phenylmethochloride (pyridinium-substituted torsemide salt)
In the same manner as in Example 3, torsemide can be reacted with benzyl chloride and triethylamine in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium phenylmethochloride.

Example 53
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium benzylthiomethochloride (pyridinium-substituted torsemide salt)
In the same manner as in Example 3, torsemide is reacted with benzyl chloromethyl thioether and triethylamine in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium benzylthiomethochloride. Can do.

Example 54
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium methylthiomethochloride (pyridinium-substituted torsemide salt)
In the same manner as Example 3, torsemide can be reacted with methyl chloromethyl thioether and triethylamine in DMF to give 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium methylthiomethochloride. it can.

Example 55
Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide mPEG350 ester)
In the same manner as in Example 3, bumetanide was reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF, where n was in the range 7-8. Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate can be obtained.

Example 56
Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide mPEG1000 ester)
In the same manner as in Example 3, bumetanide is reacted with MeO-PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF, and n is in the range of 19-24. Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate can be obtained.

Example 57
Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (bumetanide mPEG350 ester)
In the same manner as in Example 3, piretanide was reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF, where n was in the range 7-8. Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate can be obtained.

Example 58
Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate (bumetanide mPEG1000 ester)
In the same manner as in Example 3, piretanide was reacted with MeO-PEG1000-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF, and n was in the range of 19-24. Methoxy (polyethyleneoxy) _n-1 -ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate can be obtained.

Example 59
Methoxy (polyethyleneoxy) _n-1 -ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide mPEG350 ester)
In the same manner as in Example 3, furosemide is reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF, and n is in the range of 7-8. Methoxy (polyethyleneoxy) _n-1 -ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate can be obtained.

Example 60
Methoxy (polyethyleneoxy) _n-1 -ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate (furosemide mPEG1000 ester)
In the same manner as in Example 3, furosemide is reacted with MeO-PEG1000-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF, and n is in the range of 19-24. Methoxy (polyethyleneoxy) _n-1 -ethyl 5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzoate can be obtained.

Example 61
5- [1- [Methoxy (polyethyleneoxy) _n-1 -ethyl] -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide (N-mPEG350- Tetrazolyl-substituted azosemide)
In the same manner as in Example 3, azosemide is reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF, where n is in the range 7-8. 5- [1- [methoxy (polyethyleneoxy) _n-1 -ethyl] -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide is obtained Can do.

Example 62
5- [1- [Methoxy (polyethyleneoxy) _n-1 -ethyl] -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide (N-mPEG1000- Tetrazolyl-substituted azosemide)
In the same manner as in Example 3, azosemide is reacted with MeO-PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF, where n is in the range of 19-24. 5- [1- [methoxy (polyethyleneoxy) _n-1 -ethyl] -1H-tetrazol-5-yl] -2-chloro-4-[(2-thienylmethyl) amino] benzenesulfonamide is obtained Can do.

Example 63
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridiniummethoxy (polyethyleneoxy) _n-1 -ethochloride (N-mPEG350-pyridinium torsemide salt)
In the same manner as in Example 3, torsemide is reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF, where n is in the range 7-8. 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridiniummethoxy (polyethyleneoxy) _n-1 -ethochloride can be obtained.

Example 64
3-Isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridinium methoxy (polyethyleneoxy) _n-1 -ethochloride (N-mPEG1000-pyridinium torsemide salt)
In the same manner as in Example 3, torsemide is reacted with MeO-PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF, where n is in the range of 19-24. 3-isopropylcarbamylsulfonamido-4- (3′-methylphenyl) aminopyridiniummethoxy (polyethyleneoxy) _n-1 -ethochloride can be obtained.

Example 65
3-Aminosulfonyl-5-butylamino-4-phenoxybenzaldehyde (Bumetanide aldehyde)
By reacting bumetanide with bis (4-methylpiperazinyl) aluminum hydride by the method of Muraki and Mukiyama (Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215), 3-aminosulfonyl-5-butylamino -4-phenoxybenzaldehyde can be obtained.

Example 66
3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzaldehyde (pyretanide aldehyde)
Piretanide is reacted with bis (4-methylpiperazinyl) aluminum hydride by the method of Muraki and Mukiyama (Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215) to give 3-aminosulfonyl-4-phenoxy- 5- (1-Pyrrolidinyl) benzaldehyde can be obtained.

Example 67
5-Aminosulfonyl-4-chloro-2-[(2-furanylmethyl) amino] benzaldehyde (furosemide aldehyde)
Furosemide is reacted with bis (4-methylpiperazinyl) aluminum hydride by the method of Muraki and Mukiyama (Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215) to give 5-aminosulfonyl-4-chloro- 2-[(2-furanylmethyl) amino] benzaldehyde can be obtained.

Example 68
Effect of furosemide on sputum-like discharge in hippocampal slices During these studies, spontaneous sputum-like activity was induced by various doses. Sprague-Dawley rats (male and female, 25-35 days old) were decapitated, the upper surface of the skull was rapidly removed, and the brain was cooled in ice-cold oxygenated slicing medium. The slicing medium is sucrose artificial brain spinal fluid (sACSF) composed of 220 mM sucrose, 3 mM KCl, 1.25 mM NaH ₂ PO ₄ , 2 mM MgSO ₄ , 26 mM NaHCO ₃ , 2 mM CaCl ₂ and 10 mM dextrose (295 to 305 mOsm). did. The hemisphere containing the hippocampus was blocked and glued (cyanoacrylic adhesive) onto the stage of Vibroslicer (Frederick Haer, Brunsick, ME.). A 400 μm thick horizontal or transverse slice was cut into 4 ° C. oxygenated (95% O ₂ ; 5% CO ₂ ) slicing medium. Slices are quickly transferred into a holding chamber where they are oxygenated consisting of 124 mM NaCl, 3 mM KCl, 1.25 mM NaH ₂ PO ₄ , 2 mM MgSO ₄ , 26 mM NaHCO ₃ , 2 mM CaCl ₂ and 10 mM dextrose (295-305 mOsm). It was left immersed in bath medium (ACSF). Slices were kept at room temperature for at least 45 minutes before being transferred to an immersion type recording chamber (all other experiments). Within the recording chamber, slices were perfused with oxygenated recording medium at 34-35 ° C. All animal manipulations were performed according to NIH and the University of Washington animal protection guidelines.

In most slicing experiments, simultaneous extracellular field electrode recordings were obtained from the CA1 and CA3 regions. A bipolar tungsten stimulating electrode was placed on the Schaffer side branch to induce a synaptic driven electric field response at CA1. Stimulation consisted of 100-300 μsec continuous pulses with an intensity four times the population spike threshold. The post-discharge was triggered by a continuous second-like stimulus at 60 Hz for a delivery-like stimulus. Spontaneous seizure-like bursts were changed or added to the following bath media: 10 mM potassium (6 slices; 4 animals; average -81 bursts / min); 200-300 μM 4-aminopyridine (4 slices; 2 animals; average) -33 bursts / minute); 50-100 μM bicuculline (4 slices; 3 animals; average -14 bursts / minute); MGg ⁺⁺ (1 hour perfusion-3 slices; 2 animals; average -20 bursts / minute; or 3 hours perfusion) -2 slices; 2 animals); observed in slices administered with zero calcium / 6 mM KCl and 2 mM EGTA (4 slices; 3 animals). For all doses, furosemide was added to the recording medium after a certain level of burst was established.

In the first of these procedures, a post-discharge episode is induced by electrical stimulation of the Schaffer side branch (Stasheff et al., Brain Res. 344: 296, 1985) and the extracellular electric field response is induced in the CA1 pyramidal cell region. (13 slices; 8 animals). The Mg ⁺⁺ concentration in the bath medium was reduced to 0.9 mM and a post-discharge was induced by stimulation at 60 Hz for 2 seconds at an intensity 4 times the collective spike threshold (the collective spike threshold intensity was 20 for a pulse duration of 100-300 μsec. Fluctuates in the range of ~ 150 μA). The tissue was allowed to recover for 10 minutes between stimulation attempts. In each experiment, the initial response of CA1 to synaptic input was first tested by recording the electric field potential evoked by a single stimulation pulse. In the control condition, a single population spike was induced by Schaffer side branch stimulation (FIG. 1A, inset). Tonic stimulation elicited a 30 second post-discharge (Fig. 1A, left), followed by a large change in the endogenous signal (Fig. 1A, right).

  To image the intrinsic optical signal, perfusion located on the stage of an upright microscope and irradiated with white light (tungsten filament light and lens system; Dedo Inc.) directed through a microscope condenser Placed in the chamber. The light was controlled and adjusted (power supply—Lamda Inc.) to minimize fluctuations and filtered (695 nm long pass) to allow the slices to be seen at long wavelengths (red). Field of view and magnification were determined by selection of the microscope objective (4X to monitor all slices). Image frames were acquired at 30 Hz using a Charge Coupled Device (CCD) camera (Dage MTI Inc.), and Imaging Technology Inc. Using a 151 series imaging system, the 512x480 digitized 8 bits at the original spatial resolution; the gain and offset of the camera control box and A / D board were adjusted to optimize the sensitivity of the system. The imaging hardware was controlled by a 486-PC compatible computer. To increase signal / noise, an average image was created from 16 individual image frames, integrated over 0.5 seconds, and averaged together. Experimental series typically performed a continuous acquisition of a series of average images over a period of several minutes; at least 10 of these average images were acquired prior to stimulation as control images. The pseudo-colored image was calculated by subtracting the initial control image from the later acquired image and assigning a color lookup table to the pixel values. For these images, high frequency noise was typically removed using a first order low pass filter, and pixel values were mapped over the dynamic range of the system using a first order histogram stretch. All operations on these images were primary so that quantitative information was preserved. Noise is defined as the maximum standard deviation of the AR / R variation of the sequence of control images within a given acquisition series, where AR / R indicates the magnitude of the change in light transmission through the tissue. Delta R / R was calculated by taking all the difference-images and dividing by the initial control image, ie (subsequent image-initial control image) / initial control image. Noise was always <0.01 for each selected image sequence. The absolute change in light transmission through the tissue was estimated during some experiments by acquiring an image after placing a neutral density filter between the camera and the light source. On average, camera electronics and imaging system electronics are digitized after amplifying the signal by a factor of 10, so the maximum absolute change in transmission of light through the tissue was typically between 1% and 2%.

  The grayscale photograph shown in FIG. 1D is a video image of a typical hippocampal slice in the recording chamber. The fine gold wire mesh used to hold the tissue in place is observed as a dark line extending diagonally across the slice. The stimulation electrode is observed at the upper right on the CA1 ray. The recording electrode (because of the thin piece, it cannot be observed in the photograph) was inserted at the position indicated by the white arrow. FIG. 1A shows that a 2 second stimulus at 60 Hz caused post-discharge activity, showing a typical post-discharge episode recorded by the extracellular electrode. The inset of FIG. 1A shows the CA1 electric field response to a single 200 second test pulse delivered to the Schaffer side branch (arrows are artificial). FIG. 1A1 is a map of the maximum change in optical transmission through tissue induced by Schaffer side branch stimulation. The region of greatest optical change corresponds to the CA1 apex and base dendritic region on either side of the stimulation electrode. FIG. 1B is a sample chart showing the response to stimulation after 20 minutes of perfusion in a medium containing 2.5 mM furosemide. Both electrical post-discharge activity (shown in FIG. 1B) and stimulus-induced optical changes (shown in FIG. 1B1) are blocked. However, an overexcited electric field response (multiple population spike) was observed for the test pulse (inset). 1C and 1C1 show that recovery of the initial response pattern was observed 45 minutes after normal bath medium perfusion.

  The retrograde effects of furosemide block on post-stimulus discharge and the simultaneous increase in synaptic response to the test pulse have two important consequences: (1) furosemide blocks epileptiform activity and (2) synchronous (spontaneous ) And excitability (reflected by response to a single synaptic input) are dissociated. In experiments examining the dose dependence of furosemide, a minimum concentration of 1.25 mM was found to be necessary to block both post-discharge and optical changes.

Example 69
Effect of furosemide on epileptiform discharge in hippocampal slices perfused with high K ⁺ (10 mM) bath medium Rat hippocampal slices prepared as described above have long-term spontaneous seizure-like bursts with CA3 (upper chart) and CA1 (lower chart). Chart) Perfused with high K ⁺ solution until simultaneously recorded in the pyramidal cell area (FIGS. 2A and 2B). After perfusion with furosemide-containing medium (2.5 mM furosemide) for 15 minutes, the number of burst discharges increased (FIGS. 2C and 2D). However, after 45 minutes of furosemide perfusion, the burst was blocked in a reversible manner (FIGS. 2E, 2F, 2G and 2H). During the entire sequence of furosemide perfusion, the synaptic response to a single test pulse delivered to the Schaffer side branch was unchanged or enhanced (data not shown). The initial increase in the amplitude of the discharge may reflect a furosemide-induced decrease in suppression (Misgeld et al., Science 232: 1413, 1986; Thompson et al., J. Neurophysiol. 60: 105, 1988; Thompson and Gahwiller, J. Neuropysiol. 61: 512, 1989; and Pearce, Neuron 10: 189, 1993). It has been previously reported that furosemide blocks the inhibitory current component in hippocampal slices with a latency (<15 min) similar to the time to occurrence of increased excitability observed this time (Pearce, Neuron 10 189, 1993). The longer latency required for the furosemide block of spontaneous burst is thought to correspond to the additional time required for sufficient blocking of the furosemide-sensitive cell volume regulation mechanism under high K ⁺ conditions. .

After testing the effect of furosemide on slices perfused with high K ⁺ , a similar test was performed with various other commonly tested in vitro moth-like discharge models (Galvan et al., Brain Res. 241: 75, 1982; Schwartzkroin and Prince, Brain Res. 183: 61, 1980; Anderson et al., Brain Res. 398: 215, 1986; and Zhang et al., Epilepsy Res. 20: 105, 1995). After prolonged exposure (2-3 hours) to a magnesium-free medium (0-Mg ⁺⁺ ), the slices develop a wrinkle-like discharge that is resistant to commonly used anticonvulsants (Zhang et al., Epilepsy Res. 20: 105, 1995). According to records from the medial olfactory cortex (FIG. 21) and spider gyrus (not shown), 0-Mg ⁺⁺ medium perfusion 3 hours post-slice was previously described as an “anticonvulsant resistant” burst. A burst pattern with the same appearance as the one was generated. One hour after addition of furosemide to the bath medium, these bursts were blocked (FIG. 2J). Furosemide is also added / modified to the following bath media: (1) 200-300 μM 4-aminopyridine (4-AP; potassium channel blocker) added (FIGS. 2K and 2L); (2) GABA antagonist bicuculline. 50-100 μM addition (Figures 2M and 2N); (3) Magnesium removal (0-Mg ⁺⁺ )-1 hour perfusion (Figures 2O and 2P); and (4) Calcium removal + extracellular chelation (0- Spontaneous burst discharges observed by Ca ⁺⁺ ) (FIGS. 2Q and 2R) were also blocked. In these manipulations, spontaneous seizure-like patterns are recorded simultaneously from the CA1 and CA2 subfields (FIGS. 2K, 2L, 2M, and 2N display only the CA3 trace, FIGS. 2O, 2P, 2Q, And 2R display only the CA1 trace). In the 0-Ca ⁺⁺ experiment, 5 mM furosemide blocked the burst with a latency of 15-20 minutes. For all other protocols, the burst was blocked with 2.5 mM furosemide with a latency of 2-60 minutes. Furosemide reversibly blocked spontaneous burst activity in both CA1 and CA3 in all experiments (FIGS. 2L, 2N, 2P and 2R).

Example 70
The effect of furosemide on sputum-like activity induced by iv injection of kainic acid in anesthetized rats This example illustrates an in vivo model in which sputum-like activity is induced by iv injection of kainic acid (KA) to anesthetized rats. (Lothman et al., Neurology 31: 806, 1981). The results are shown in FIGS. Sprague-Dawley rats (4 animals; body weight 250-270 g) are anesthetized with urethane (1.25 g / kg ip) and further urethane injections (0.25 g / kg ip) as required. To maintain anesthesia. Body temperature was monitored using a rectal temperature probe and maintained at 35-37 ° C. with a heating pad; heart rate (EKG) was continuously monitored. The jugular vein was intubated on one side for intravenous drug administration. Rats are placed in a Kopf stereotaxic device (with the top of the height of the skull) and a bipolar stainless steel microelectrode insulated to 0.5 mm apex is inserted from the cortical surface to a depth of 0.5-1.2 mm EEG rhythm (EEG) activity in the frontal parietal cortex was recorded. In some experiments, hippocampal EEG was recorded by raising a 2M NaCl containing pipette to a depth of 2.5-3.0 mm. Data was stored on VHS videotape and analyzed offline.

  After the surgical procedure and electrode placement, the animals were allowed to recover for 30 minutes before starting an experiment with kainate injection (10-12 mg / kg iv). Strong seizure activity, increased heart rate and rapid nasal hair movement were induced with a latency of about 30 minutes. After stable electrical seizures became apparent, furosemide was delivered at 20 mg / kg instantaneous injections every 30 minutes until a total of 3 injections. The experiment was terminated by intravenous administration of urethane. Animal protection was approved by the University of Washington Animal Protection Committee in accordance with NIH guidelines.

  FIGS. 3A-3H show kainic acid-induced electrical “convulsive status” furosemide block in urethane anesthetized rats. The EKG recording is shown in the upper chart, and the EEG recording is shown in the lower chart. In this model, a strong electrical discharge (electrical “convulsive status”) was recorded from the cortex (or from the deep hippocampal electrode) 30-60 minutes after KA injection (10-12 mg / kg). 3C and 3D). Control experiments (and previous reports, Lothman et al., Neurology, 31: 806, 1981) show that this status-like activity is well maintained over 3 hours. Subsequent intravenous injection of furosemide (cumulative dose: 40-60 mg / kg) blocked seizure activity at a latency of 30-45 minutes and often resulted in relatively flat EEG (FIGS. 3E, 3F, 3G and 3H). Even 90 minutes after furosemide injection, cortical activity maintained near normal baseline levels (ie, those observed prior to KA and furosemide injection). Studies on the pharmacokinetics of furosemide in rats have shown that the dose used in this example is well below the level of toxicity (Hammarlund and Paalzow, Biopharmaceutics Drug Disposition, 3: 345, 1982).

Experimental Methods for Examples 71-74 Hippocampal slices were prepared from Sprague-Dawley adult rats as described above. A transverse hippocampal slice having a thickness of 100 μm was cut out with a vibration cutter. Slices typically contained the entire hippocampus and spider gyrus. After cutting, the slices were stored in an oxygenated holding chamber at room temperature for at least 1 hour before recording. All recordings were acquired in a chamber of interface type with oxygen added at _{34~35 ℃ (95% O 2,} 5% CO 2) artificial cerebrospinal fluid (ACSF). Normal ACSF contained (in mmol / l units) 124 NaCl, 3 KCl, 1.25 NaH ₂ PO ₄ , 1.2 MgSO ₄ , 26 NaHCO ₃ , 2CaCl ₂ and 10 dextrose.

  A Sharp electrode for intracellular recording from CA1 and CA3 pyramidal cells was filled with 4M potassium acetate. Electric field recordings from the CA1 and CA3 cell body layers were obtained using low resistance glass electrodes filled with 2M NaCl. A small monopolar tungsten electrode was placed on the slice surface for stimulation of the Schaffer side branch or hilar pathway. Spontaneous and stimulus-induced activities from electric fields and intracellular recordings were digitized (Neurocoder, Neurodata Instruments, New York, NY) and stored on videotape. Offline analysis of the data was performed using AxoScope software (Axon Instruments) on a personal computer.

In some experiments, normal or low chloride media containing bicuculline (20 μM), 4-aminopyridine (4-AP) (100 μM) or high K ⁺ (7.5 or 12 mM) was used. Low chloride solutions (7 and 21 mM [Cl ⁻ ] _o ) were prepared by equimolar replacement of NaCl and Na ⁺ -gluconate (Sigma) in all experiments. All solutions were prepared so that they had a pH of 7.4 and an osmotic pressure of 290-300 mOsm at about 35 ° C. and in equilibrium with carbonation with 95% O ₂ /5% CO ₂ .

  After being placed in the interface chamber, the slice was surface washed at approximately 1 ml / min. At this low flow rate, it took 8-10 minutes to complete the perfusion medium exchange. All times reported here account for this delay and have an error of about ± 2 minutes.

Example 71
Timing of termination of spontaneous wrinkle-like bursts within CA1 and CA3 The relative contribution of factors that modulate synchronous activity differs between CA1 and CA3. These factors include regional circulation differences and region-specific differences in cell filling and volume ratio of extracellular space. Given that the antidepressant effect of anion or chloride cotransport antagonists is due to desynchronization at the timing of neuronal discharge, the chloride cotransport block is predicted to differentially affect the CA1 and CA3 regions. To test this, a series of experiments were performed to characterize the difference in the timing of spontaneous sputum-like activity blocks in the CA1 and CA3 regions.

Electric field activity is recorded simultaneously in the CA1 and CA3 regions (approximately the midpoint between the proximal and distal of the CA3 region) and spontaneous bursts are high-[K ⁺ ] _o (12 μM; n = 12), It was induced by administration of bicuculline (20 mM; n = 12) or 4-AP (100 μM; n = 5). A single electrical stimulus was delivered to the Schaffer side branch in the middle between the CA1 and CA3 zones every 30 seconds so that the electric field responses in the CA1 and CA3 zones were monitored throughout the duration of each experiment. In all experiments, a continuous spontaneous wrinkle-like burst was observed for at least 20 minutes prior to switching to low- [Cl ⁻ ] _o (21 mM) or furosemide-containing (2.5 mM) medium.

  In all cases, after 30-40 minutes exposure to furosemide or low chloride media, spontaneous bursts were stopped in CA1 before the burst stopped in CA3. The temporal sequence of events typically observed included an initial increase in burst frequency and amplitude of spontaneous electric field events, followed by a decrease in burst discharge amplitude that was more rapid in CA1 than in CA3. After CA1 became silent, CA3 continued to discharge for 5-10 minutes until it also showed no spontaneous wrinkle-like events.

The temporal pattern of the burst stop, low or agents used to block the spontaneous burst is furosemide - [Cl ^-] Regardless of whether the _o medium, observed in all epileptiform induction dose tested It was done. Throughout all stages of these experiments, Schaffer side branch stimulation induced an overexcited electric field response in both the CA1 and CA3 cell body layers. Immediately after spontaneous bursts were blocked in both the CA1 and CA3 zones, hyperexcited population spikes could still be triggered.

  The inventors have observed that the cessation of burst in CA1 before CA3 is an anthropogenic factor in the organization of synaptic contacts between these areas when compared to our selection of recording sites I thought. Projection of the various subregions of CA3 is known to terminate in an organized manner in CA1; CA3 cells that are closer to the dentate gyrus (proximal CA3) and the distal portion of the CA1 (proximal gyrus) CA3 projections that result from cells that are located more proximally in CA3 end to a greater extent in the portion of CA1 that is located closer to the CA2 boundary. If burst arrest occurs in different sub-regions of CA3 at different times, the results of the above experimental set will not occur as a difference between CA1 and CA3, but as a function of burst activity variability across the CA3 sub-region. Conceivable. We tested this possibility in three experiments. Immediately after the spontaneous burst stopped in CA1, we examined the CA3 electric field using the recording electrode. Records from several different CA3 positions (the most proximal to the most distal part of CA3) indicated that all subregions of the CA3 region were spontaneously bursting during the time when CA1 was silent.

  The observation that CA3 continued to discharge spontaneously after CA1 became silent is that the collective discharge in CA3 is generally thought to induce discharge in CA1 via excitatory synaptic transmission. It was unexpected. As noted above, single pulse stimulation delivered to the Schaffer side branch still induces multiple population spikes in CA1 even after blocking of spontaneous bursts; ie, excitatory synapses of hyperexcitation from CA3 to CA1 Transmission was undamaged. Assuming that synaptic transmission and sustained spontaneous electric field discharge maintained in this way exist in CA3, we believe that loss of spontaneous burst in CA1 reduces the synchronization of the excitatory driving force applied. Presumed to be due. Furthermore, since the spontaneous soot-like discharge in CA3 eventually stops, it is considered that this desynchronization process occurred at different times in the two hippocampal sub-electric fields.

Example 72
Effect of chloride co-transport antagonism on the synchronization of CA1 and CA3 electric field collective discharges The observations of Example 4 show that the temporal relationship between exposure time to low- [Cl ⁻ ] _o or furosemide-containing media and spontaneous burst activity characteristics Suggested. Furthermore, this relationship was different between the CA1 and CA3 regions. To further clarify the temporal relationship, we compared the occurrence of CA1 action potentials with population spike events in the CA1 and CA3 electric field responses during spontaneous and stimulus-induced burst discharges.

Intracellular recordings were obtained from CA1 pyramidal cells using an intracellular electrode placed in close proximity (<100 μM) to the CA1 field electrode. Slices were stimulated every 20 seconds using a single stimulus delivered to the shuffler side branch. After a sustained spontaneous burst was established for at least 20 minutes, the bath medium was switched to bicuculline-containing low- [Cl ⁻ ] _o (21 mM) medium. After about 20 minutes, the burst frequency and amplitude reached their maximum values. The simultaneous electric field and intracellular recording during this time indicated that the CA1 electric field and intracellular recording were closely synchronized with the CA2 electric field discharge. During each spontaneous discharge, the response of the CA3 electric field was several milliseconds ahead of the CA1 discharge. During the stimulation-evoked event, the action potential discharge of CA1 pyramidal cells was closely synchronized with both the CA3 and CA1 field discharges.

Low - [Cl ^-] were exposed continuously to _o medium, the latency between the spontaneous discharge of CA1 and CA3 region increases, the maximum latency 30-40 ms exposure to bicuculline-containing low chloride medium It happened 30-40 minutes later. During this time, the amplitude of the spontaneous electric field discharge of CA1 and CA3 therapy decreased. This time-stimulated discharge closely mimics the morphological characteristics and the spontaneously occurring discharge in relative latency. However, initial stimulation-induced depolarization of neurons (perhaps a single synaptic EPSP) began without any significant increase in latency. The time interval at which these data are acquired corresponds to the time immediately before the stop of the spontaneous burst in CA1.

Low - ^[Cl _-] after perfusion 40-50 minutes using _o medium, spontaneous burst has been lost almost in CA1, had no effect in CA3. This time of Schaffer side branch stimulation indicates that the single-synaptic trigger response of CA1 pyramidal cells occurred without any significant increase in latency, but the stimulus-induced electric field response almost disappeared. The time interval at which these data are acquired corresponds to the time immediately before the stop of the spontaneous burst in CA3.

After prolonged exposure to low- [Cl ⁻ ] _o medium, a significant increase (> 30 ms) occurred in the latency between Schaffer side branch stimulation and the resulting CA3 field discharge. Eventually, there was no electric field response induced by the Schaffer side branch stimulation in both the CA1 and CA3 regions. However, action potential discharge from CA1 pyramidal cells in response to the Schaffer side branch was elicited with little change in response latency. In fact, for the entire duration of the experiment (greater than 2 hours), action potential discharge from CA1 pyramidal cells was induced with short latency by Schaffer side branch stimulation. Furthermore, the stimulation-induced hyperexcitatory discharge of CA3 was eventually blocked after prolonged exposure to low- [Cl ⁻ ] _o medium, but the retrograde response in CA3 was observed to be preserved. .

Example 73
Effect of chloride co-transport antagonism on burst discharge synchronization in CA1 pyramidal cells The above data indicate that loss of electric field response may be due to desynchronization of action potential generation between neurons. That is, synaptic drive excitation of CA1 pyramidal cells was not preserved, but the synchronism of action potentials within the CA1 neuron population was not sufficient to add up to a measurable DC electric field response. To test this, paired intracellular records of CA1 pyramidal cells were acquired simultaneously with the CA1 electric field response. In these experiments, both the intracellular electrode and the electric field recording electrode were placed within 200 μm of each other.

Bicuculline-containing low ^- [Cl -] _o medium by periods of induced maximum spontaneous activity, the recording is firmly action potentials between AC1 neurons and CA1 field discharges pair both during spontaneous and stimulation-evoked discharges Indicates that it was synchronized. After continuous exposure to low- [Cl ⁻ ] _o medium, both spontaneous and stimulated evoked discharges of action potentials between pairs of CA1 neurons at the time when the amplitude of the CA1 electric field discharge begins to broaden and decay. Desynchronization was shown at the time of occurrence and between action potential and electric field response. Desynchronization was consistent with suppression of CA1 field amplification. By the time the spontaneous burst in CA1 ceased, there was a significant increase in latency between the Schaffer side branch stimulus and the CA1 electric field discharge. At this point, paired intracellular recordings showed dramatic desynchronization at the timing of action potential discharges between neuron pairs and between action potential generation and Schaffer side branch-stimulated electric field discharges.

  The observed desynchronization of CA1 action potential discharge may be due to disruption of the timing of synaptic release or the randomization of the mechanisms necessary for synaptic drive action potential generation such as random conduction failure in neuronal processes There is sex. In that case, it is predicted that the generation of action potentials between a predetermined neuron pair varies very randomly with respect to each other for each stimulus. We tested this by comparing the action potential discharge pattern of the neuron pair during multiple consecutive stimuli of the Schaffer side branch. During each stimulation event, action potentials occurred at approximately the same time relative to each other, showing approximately the same burst morphological characteristics for each stimulation. We also investigated whether the generation of action potentials during a given pair of neurons during a spontaneous electric field discharge is fixed in time. The pattern of action potential discharges from a given pair of CA1 neurons was compared between successive spontaneous electric field bursts of time when action potential generation was clearly desynchronized. As in the case of the stimulation-evoked action potential discharge described above, the action potentials generated during the spontaneous collective discharge occur at almost the same time relative to each other, and almost the same burst in one spontaneous discharge and the next Of morphological features were observed.

Example 74
Effects of low chloride administration on spontaneous synaptic activity The antidepressant effects associated with chloride co-transport antagonism may be mediated by some effects of transmitter release. Blocking chloride co-transport can alter the amount and timing of transmitters released from the terminus, thereby affecting neuronal synchronization. To investigate whether low- [Cl ⁻ ] _o exposure affects the mechanisms associated with transmitter release, cells during administration that dramatically increase the spontaneous synaptic release of transmitter from the presynaptic terminal The internal CA1 response was recorded simultaneously with the CA1 and CA3 electric field responses.

Increased spontaneous release of the transmitter was induced by 4-AP (100 μM) administration. After 40 minutes exposure to 4-AP containing medium, spontaneous synchronous burst discharges were recorded in the CA1 and CA3 areas. Switching to 4-AP containing low- [Cl ⁻ ] _o medium enhanced spontaneous bursts as initially observed. High gain intracellular recordings indicated that high amplitude spontaneous synaptic activity was induced by 4-AP administration. Further exposure to low chloride media blocked burst discharge in CA1, but CA3 continued to discharge spontaneously. At this point, the CA1 intracellular record was further increased in spontaneous synaptic noise and maintained its state during prolonged exposure to 4-AP containing low chloride media. These data suggest that the mechanism responsible for synaptic release from the terminal is not adversely affected by low chloride exposure in an embodiment that accounts for the block of 4-AP-induced spontaneous burst in CA1. These results also eliminate the possibility that the effects of low- [Cl ⁻ ] _o exposure are due to alterations in CA1 dendritic properties that sacrifice their efficiency of conducting PSPs to the body.
Experimental Methods for Examples 75-79 In all of the following experiments, [Cl ⁻ ] _o was reduced by equimolar replacement of NaCl and Na ⁺ -gluconate. Gluconate was used rather than other anion exchanges for several reasons. First, patch clamp testing suggests that gluconate is substantially impermeable to chloride channels while other anions (eg, sulfate, isethionate and acetate) are permeable to varying degrees. I understood it. Secondly, transport of extracellular potassium via glial NKCC1 cotransport is blocked when extracellular chloride is replaced by gluconate but not completely when it is replaced by isethionate. Since this furosemide-sensitive cotransporter plays a prominent role in cell swelling and volume change of the extracellular space (ECS), the effect of our administration has been shown to be a previous furosemide experiment (Hochman et al. Science, 270: 99). -102, 1995; US Pat. No. 5,902, 732) we wished to use a suitable anion replacement. Third, formate, acetate and propionate generate weak acids when used as Cl ^- substitutes, resulting in a rapid drop in intracellular pH; gluconate remains extracellular and is reported to induce intracellular pH fluctuations. It has not been. Fourth, for comparative purposes, we will use the same anion replacement as used in previous studies to investigate the effect of low- [Cl ⁻ ] _{o on} ECS activity-induced changes. I hoped.

There are some suggestions that replacement of certain anions chelate calcium. Later studies have failed to clarify any significant ability of anion substitution to chelate calcium, but this issue is still a concern in the literature. Calcium chelate formation is a value of several hours of exposure to media in which the resting membrane potential is maintained normally and the [Cl ⁻ ] _o is reduced by gluconate substitution in the following experiments: However, the synaptic response (actually overexcited synaptic response) can occur, so it was considered not to be a problem. Furthermore, we have found that the calcium concentration in our low- [Cl ⁻ ] _o medium is the same as that in our control medium measured using a Ca ²⁺ selective microelectrode. I have confirmed.

Sprague-Dawley adult rats were prepared as described above. In other words, a transverse hippocampal slice having a thickness of 400 μm was cut out with a vibration cutter. Slices typically contained the entire hippocampus and spider gyrus. After cutting, the slice was stored in an oxygenated holding chamber for at least 1 hour before recording. All recordings were acquired in a chamber of interface type with oxygen added at _{34~35 ℃ (95% O 2,} 5% CO 2) artificial cerebrospinal fluid (ACSF). Normal ACSF contained (in mmol / l units) 124 NaCl, 3 KCl, 1.25 NaH ₂ PO ₄ , 1.2 MgSO ₄ , 26 NaHCO ₃ , 2CaCl ₂ and 10 dextrose. In some experiments, normal or low chloride media containing bicuculline (20 μM), 4-AP (100 μM) or high K ⁺ (12 mM) was used. Low chloride solutions (7, 16 and 21 mM [Cl ⁻ ] _o ) were prepared by equimolar replacement of NaCl and Na ⁺ -gluconate (Sigma Chemical Co., St. Louis, Mo.). All solutions were prepared so that they had a pH of 7.4 and an osmotic pressure of 290-300 mOsm at about 35 ° C. and in equilibrium with carbonation with 95% O ₂ /5% CO ₂ .

  For intracellular recording from CA1 pyramidal cells, a Sharp electrode filled with 4M potassium acetate was used. Recording of the electric field from the CA1 or CA3 cell body layer was obtained using a low resistance glass electrode filled with NaCl (2M). For stimulation of the Schaffer side branch pathway, a small monopolar electrode was placed on the slice surface in the middle between the CA1 and CA3 zones. Spontaneous and stimulus-induced activities from electric fields and intracellular recordings were digitized (Neurocoder, Neurodata Instruments, New York, NY) and stored on videotape. Offline analysis of the data was performed using AxoScope software (Axon Instruments) on a PC-computer.

Ion selective microelectrodes were made according to standard methods well known in the art. The pipette of the double barrel was stretched and cut so as to be a straight line of about 3.0 μm. The control barrel was filled with ACSF and the other barrel was silanized and the tip was backfilled with a resin selective for K ⁺ (Corning 477317). The remainder of the silane treatment barrel was filled with KCl (140 mL). Each barrel was introduced into a high impedance dual differential amplifier (WPIFD 223) via an Ag / AgCl wire. Each ion-selective microelectrode was calibrated with a solution of known ionic composition and considered appropriate if it was characterized by a nearly Nernst-type slope response and remained stable throughout the experiment. .

  After being placed in the interface chamber, the slice was surface washed at approximately 1 ml / min. At this low flow rate, it took 8-10 minutes to complete the perfusion medium exchange. All times reported here account for this delay and have an error of about ± 2 minutes.

Example 75
[Cl ^- -] Low cortical and hippocampal slices according to the action other tests of _o - [Cl ^-] Exposure to _o are basic intrinsic and synaptic features, for example, the input resistance, low against the CA1 field recording It has been shown that it does not affect quiescent membrane potential, depolarization-induced action potential generation or excitatory synaptic transmission. Low to CA1 area of the previous studies also hippocampus - [Cl ^-] The epileptogenic properties of _o exposure are partially characterized. The following test low - [Cl ^-] were performed to observe the _o-induced hyperexcitability and excess synchronization start and the thought can stop time. Slices (n = 6) were first perfused with normal media until stable intracellular and electric field recordings were established in CA1 pyramidal cells and CA1 cell body layers, respectively. In two experiments, the same cells were retained throughout the experiment (> 2 hours). In the remaining experiments (n = 4), the initial intracellular record disappeared with the medium change and additional records were obtained from different cells. The pattern of neuronal activity in these experiments was identical to that observed when single cells were observed.

The electric field and intracellular electrode were always placed close to each other (<200 μm). In each case, spontaneous bursts occurred first at the cellular level and then at the electric field level after approximately 15-20 minutes exposure to low- [Cl ⁻ ] _o medium (7 mM). This spontaneous electric field activity is indicative of a synchronous burst discharge in a large population of neurons, and after continuing for 5-10 minutes, the electric field recording became silent. When the electric field first became silent, the cells continued to discharge spontaneously. This result suggests that although the collective activity was “desynchronized”, the cells' discharge ability was not impaired. After about 30 minutes of exposure to low- [Cl ⁻ ] _o medium, intracellular recordings indicate that the cells continued to discharge spontaneously despite the electric field remaining silent. Yes. Response of the cell to intracellular current injection at 2 time points, the ability of cells to generate an action potential is low ^- [Cl -] _o indicates that has not been impaired by exposure. Furthermore, electrical stimulation in the CA1 radiation layer caused a burst discharge, indicating that an overexcited state was maintained in the tissue.

Example 76
Low for ^[K _{+] o-induced} epileptiform activity - - High in CA1 ^[Cl _-] previous set of _o effects experimental low - ^[Cl _-] tissue exposure to _o medium short stop within 10 minutes It has been shown that induced a potential burst of spontaneous electric field. If the decrease in [Cl ⁻ ] _o can eventually eventually block spontaneous trap-like (ie, synchronous) bursts, these results indicate that after the burst activity of this initial period has stopped Only suggests that the anti-depressive effect is observable. Accordingly, the present inventors have high - examined the temporal effect of _o administration low for ^[K _{+] o-induced} burst activity - - _^[Cl]. Slices (n = 12) were exposed to medium in which [K ⁺ ] _o had been raised to 12 mM, and the electric field potential was recorded using the electric field electrode in the CA1 cell body layer. Spontaneous field potential bursts were observed for at least 20 minutes, and then the slices were exposed to media in which [K ⁺ ] _o was maintained at 12 mM while [Cl ⁻ ] _o was reduced to 21 mM. Within 15-20 minutes after exposing the tissue to low- [Cl ⁻ ] _o / high- [K ⁺ ] _o medium, the burst amplitude increased and each electric field event had a longer duration. . After this brief accelerated field activity (lasting 5-10 minutes), the burst stopped. To test whether this block is reversible, after the electric field potential has been silent for at least 10 minutes, we have returned to normal [Cl ⁻ ] _o high- [K ⁺ ] _o medium. It was. The burst returned within 20-40 minutes. Throughout each experiment, the CA1 electric field response to Schaffer side branch stimulation was monitored. The maximum electric field response was recorded just before the spontaneous burst stopped, during the period when the spontaneous burst had the maximum amplitude. However, even after blocking spontaneous bursts, multiple population spikes were evoked by Schaffer side branch stimulation, indicating that synaptic transmission was intact and that the tissue remained overexcited.

In 4 slices, intracellular recordings of CA1 pyramidal cells were acquired along with CA1 electric field recordings. During the period of high- [K ⁺ ] _o induced spontaneous burst, hyperpolarized current was injected into the cells so that the post-synaptic potential (PSP) could be observed well. Spontaneously generated action potentials and PSP were still observed after a low- [Cl ⁻ ] _o block of spontaneous burst. These observations further support the view that synaptic activity itself was not blocked by low- [Cl ⁻ ] _o administration.

Example 77
4-AP, high- [K ⁺ ] _o and bicuculline-induced low- [Cl ⁻ ] _o block in CA1 and CA3 We next show that we are about furosemide administration as the epileptiform activity in the CA1 and CA3 region was caused by different pharmacological administration low - to test whether _o administration can block ^- [Cl]. For this experimental set, we were able to use high- [K ⁺ ] _o (12 μM) (n = 5), 4-AP (100 μM) (n = 4) and bicuculline (20 and 100 μM) (n = 5). low on spontaneous burst had been induced - [Cl ^-] was chosen to test the effect of _o administration. In each set of experiments, the electric field response was recorded spontaneously from the CA1 and CA3 zones, and in each case, CA1 and CA3 within 30 minutes after reducing [Cl ⁻ ] _o in the perfusion medium to 21 mM. Spontaneous sputum-like activity in both areas was reversibly blocked. These data suggest that, like furosemide, low- [Cl ⁻ ] _o reversibly blocks spontaneous bursts in some of the most commonly studied in vivo models of epileptiform activity. ing.

Example 78
High - ^[K _+] low for a block of _o-induced epileptiform activity - ^[Cl _-] The data obtained from the set of experiments before comparison between the _o and furosemide, low - ^[Cl _-] of both _o and furosemide It is consistent with the hypothesis that antiepileptic effects are mediated by their effects on the same physiological mechanism. To further examine this hypothesis, we recorded as low- [Cl ⁻ ] _o (n = 12) and furosemide (2) for high- [K ⁺ ] _o induced bursts as records using the electric field electrode in CA1. The temporal sequence of action of .5 and 5 mM) (n = 4) was compared. This indicates that administration of both low- [Cl ⁻ ] _o and furosemide induced a similar temporal sequence of action, namely the initial short time electric field activity amplification amplitude and subsequent spontaneous field activity block (reversible). all right. In both cases, electrical stimulation of the Schaffer side branch caused an overexcited response even after the spontaneous burst was blocked.

Example 79
Results of extended exposure to low- [Cl ⁻ ] _o medium containing variable [K ⁺ ] _{o In} the experiments described above, we have not been able to block spontaneous bursts by low- [Cl ⁻ ] _o exposure. The electric field activity in some slices was monitored over 1 hour. After such prolonged exposure, a spontaneous long-lasting depolarization shift occurred. The morphological characteristics and frequency of these post power plant events were thought to be related to extracellular potassium and chloride concentrations. Because of these observations, we have performed an experimental set of systematically varying [Cl ⁻ ] _o and [K ⁺ ] _o to determine the extent of these ions for subsequent spontaneous electric field events. The effect of variation was observed.

In our first set of experiments, slices were exposed to medium containing low [Cl ⁻ ] _o (7 mM) and normal [K ⁺ ] _o (3 mM) (n = 6). After exposure to this medium for 50-70 minutes, spontaneous events were recorded in the CA1 area; these events occurred as a 5-10 mV negative shift in the DC field and the first episode lasted 30-60 seconds. Each subsequent episode was longer than the previous one. This observation suggests that the non-homeostasis mechanism decayed over time as a result of the ionic concentration in the bath medium. In some experiments (n = 2) in which these negative DC field shifts were induced, intracellular recordings of CA1 pyramidal cells were acquired simultaneously with the CA1 field recording.
For these experiments, intracellular and electric field records were obtained in close proximity to each other (<200 μm). Prior to each negative field shift (10-20 seconds), the neurons began to depolarize. Cell depolarization was indicated by a decrease in resting membrane potential, increased spontaneous firing frequency, and decreased action potential amplitude. Consistent with the onset of the negative electric field shift, the cells were sufficiently depolarized to fail to fire spontaneous or current-induced (not explained) action potentials. Since the neuronal debunker started 10-20 seconds before the electric field shift, the increase in extracellular potassium over time resulted in depolarization of the neuronal population, which is thought to initiate these electric field events. Such an increase in [K ⁺ ] _o is thought to be due to alteration of the chloride-dependent glial co-transport mechanism that normally moves potassium from the extracellular to the intracellular space. To [K ^+] _o elevated test whether preceded it such negative field shifts (and equilibrated cell depolarization), K + ^- changes in using selective microelectrode [K ^+] _o The recorded experiment (n = 2) was performed.

In each experiment, K ⁺ -selective microelectrodes and electric field electrodes were placed in close proximity to each other (<200 μm) in the CA1 cone layer and stimulated to the shuffler side branch every 20 seconds so that the population spike size could be monitored A pulse was delivered. Multiple spontaneously generated negative field shifts were induced by perfusion with low- [Cl ⁻ ] _o (7 mM) medium. Each event is accompanied by significant increase in [K ^+] _o, increase in [K ^+] _o was started a few seconds before the negative electric field shift start. A slow 1.5-2 mM [K ⁺ ] _o increase occurred over a time interval of approximately 1-2 minutes before the start of each event. The stimulus-induced electric field response gradually increased in amplitude over time with an increase in [K ⁺ ] _o until just before the negative electric field shift.

In the second experimental set (n = 4), [K ⁺ ] _o was raised to 12 mM and [Cl ⁻ ] _o was raised to 16 mM. After exposure to this medium for 50-90 minutes, slow oscillations were recorded in the CA1 region. These oscillations were characterized by a negative DC shift of 5-10 mV of electric field potential and had a periodicity of about 1 cycle / 40 seconds. Initially, these vibrations occurred completely and had an irregular shape. Over time, these vibrations became continuous and produced a regular waveform. With exposure to furosemide (2.5 mM), the amplitude of vibration gradually decreased and the frequency increased until the vibration was completely blocked. Such low in this like slices ^- [Cl -] _o-induced vibrations has not been reported previously. However, the temporal characteristics of the vibration event were very similar to the low- [Cl ⁻ ] _o induced [K ⁺ ] _o vibrations previously reported purely in axonal preparations.

In the third set of experiments (n = 5), [Cl ⁻ ] _o was further increased to 21 mM and [K ⁺ ] _o was reduced again to 3 mM. In these experiments, a single infrequent negative field potential shift occurred within 40-70 minutes (data not shown). These events (5-10 mV) lasted 40-60 seconds, occurred at random intervals, and maintained a relatively constant duration throughout the experiment. These events may be triggered by a single electrical stimulation delivered to the Schaffer side branch.

Finally, in the final experimental set (n = 5), [Cl ⁻ ] _o was further increased to 21 mM and [K ⁺ ] _o was increased to 12 mM. In these experiments, late spontaneous electric field events were not observed during the course of the experiment (2-3 hours).

Example 80
Changes in [K ⁺ ] _o during low chloride exposure Sprague-Dawley adult rats were prepared as described above. A transversal hippocampal slice having a thickness of 400 μm was cut out with a vibration cutter and stored in an oxygenated holding chamber for 1 hour before recording. An immersion chamber was used for K ⁺ selective microelectrode recording. Slices were perfused with oxygenated at _{34~35 ℃ (95% O 2,} 5% CO 2) artificial cerebrospinal fluid (ACSF). Normal ACSF contained 10 mM dextrose, 124 mM NaCl, 3 mM KCl, 1.25 mM NaH ₂ PO ₄ , 1.2 mM MgSO ₄ , 26 mM NaHCO ₃ and 2 mM CaCl ₂ . In some experiments, normal or low chloride media containing 4-aminopyridine (4-AP) at 100 μM was used. A low chloride solution (21 mM [Cl ⁻ ] _o ) was prepared by equimolar replacement of NaCl and Na ⁺ -gluconate (Sigma Chemical Co.).

  Recording of the electric field from the CA1 or CA3 cell body layer was obtained using a low resistance glass electrode filled with NaCl (2M). For stimulation of the shuffler side branch pathway, a monopolar stainless steel electrode was placed on the slide surface in the middle between the CA1 and CA3 zones. All records were digitized (Neurocoder, Neurodata Instruments, New York, NY) and stored on videotape.

K ⁺ -selective microelectrodes were made according to standard methods. In other words, the control barrel of a double barrel pipette was filled with ACSF, and the other barrel was silanized and the tip was backfilled with KCl with K ⁺ selective resin (Corning 477317). An ion selective microelectrode was calibrated and considered appropriate if it had a nearly Nernst-type slope response and remained stable throughout the experimental period.

Low - [Cl ^-] Exposure of hippocampal slices in the _o medium has been found to encompass a time-dependent sequence of changes on the activity of CA1 pyramidal cells, it involves three distinct steps described above. Briefly, low - [Cl ^-] hyperexcitability and spontaneous epileptiform discharge and brief increase exposure to _o the medium occurs. Further exposure to low- [Cl ⁻ ] _o medium blocks spontaneous sputum-like activity, but intracellular hyperexcitation remains, and action potential firing times are less synchronized with each other. Eventually, with prolonged exposure, the action potential firing time is sufficiently desynchronized so that the stimulus-evoked electric field response completely disappears, yet individual cells continue to exhibit a single synapse-evoked response to the Schaffer side branch stimulus. The following results show that the anticonvulsant effect of furosemide on chloride co-transport antagonism is independent of its direct effect on excitatory synaptic transmission and desynchronization of population activity with any of our accompanying excitatory declines Is the result of

In 6 hippocampal slices, K ⁺ selective electric field microelectrodes were placed in the CA1 cell body layer, stimulation electrodes were placed in the Schaffer side branch pathway, and single pulse stimulation (300 μs) was delivered every 20 seconds. After a stable baseline [K ⁺ ] _o was obtained for at least 20 minutes, perfusion was switched to low- [Cl ⁻ ] _o medium. The electric field response became hyperexcitable as the increase in [K ⁺ ] _o began within 1-2 minutes of exposure to low- [Cl ⁻ ] _o medium. After approximately 4-5 minutes of low- [Cl ⁻ ] _o medium exposure, the magnitude of the electric field response decayed and disappeared completely. The corresponding record for [K ⁺ ] _o starts to increase immediately after potassium exposure to low- [Cl ⁻ ] _o medium, and this maximum [K ⁺ ] _o rise corresponds to the maximum overexcited CA1 electric field response in time Was. Consistent with the decrease in magnitude of the electric field response, [K ⁺ ] _o begins to decay until 8-10 minutes exposure to low- [Cl ⁻ ] _o medium exposure, and for the remainder of the experiment It was constant at higher values than the 1.8-2.5 mM control level. Four slices were returned to control medium and allowed to fully recover. The experiment was then repeated using K ⁺ selective microelectrodes placed in the radiation. Similar sequence changes of [K ⁺ ] _o were observed in the dendritic layer, with values of [K ⁺ ] _o being 0.2-0.3 mM lower than those observed in the cell body layer.

In 4 hippocampal slices, the response of stimulation-induced changes in [K ⁺ ] _o was compared between the control state and the CA1 electric field response completely disappeared by low- [Cl ⁻ ] _o medium exposure. In each slice, [K ⁺ ] _o selectivity measurements were first acquired in the cell body layer and then fully recovered in control medium and then experimented with K ⁺ selective electrodes that were transferred to the ray. Repeated. Each stimulation trial consisted of a 10 Hz burst delivered to the Schaffer side branch for 5 seconds. Maximum increase in [K ^+] _o the control conditions and the low - [Cl ^-] between the _post-o medium extended exposure and were similar between the cell body and dendritic layers. However, the recovery time observed after low- [Cl ⁻ ] _o medium extended exposure was significantly longer than that observed during control conditions.

These results indicate that furosemide administration resulted in increased [K ⁺ ] _o in the extracellular space. Exposure of brain tissue to low- [Cl ⁻ ] _o media immediately induces an increase of 1-2 mM [K ⁺ ] _o that is maintained throughout the duration of exposure, with an initial increase in excitability and CA1 It was consistent with the final disappearance of the electric field response. This loss of CA1 electric field response during low- [Cl ⁻ ] _o exposure is most likely due to neuronal firing time desynchronization. Importantly, the stimulation-induced elevation of [K ⁺ ] _o in both the cell body and the dendritic layer was nearly identical before and after the completely low- [Cl ⁻ ] _o block of the CA1 electric field response. This data suggests that comparable stimulus-induced synaptic drive and action potential generation occurred under control conditions and after low- [Cl ⁻ ] _o blocking of the electric field. Taken together, these data indicate that the anti-depressant and desynchronizing effects of the chloride cotransport antagonist furosemide are independent of direct effects on excitatory synaptic transmission, and the resynchronization of population activity without reduced excitability. The result is shown.

Example 81
Low chloride changes furosemide and low extracellular pH in exposure - [Cl ^-] in the extracellular pH changes anion / antagonists of chloride-dependent cotransporter that may contribute to the maintenance of synchronous population activity, such as _o May affect. Rat hippocampal brain slices were prepared as described in Example 80 except that NaHCO ₃ was replaced with an equimolar amount of HEPES (26 nM) and an interfaced chamber was used.

In 4 hippocampal brain slices, continuous spontaneous burst was induced by exposure to medium containing 100 μM 4-AP as described in Example 13. Electric field recordings were acquired simultaneously from the cell body layer in the CA1 and CA3 regions. Stimulation was delivered every 30 seconds to the Schaffer side branch throughout the experiment. After continuous bursts were observed for at least 20 minutes, the slices were exposed to 4-AP-containing HEPES medium that was nominally free of bicarbonate. As a result of prolonged exposure to HEPES medium (0.2 hours), no significant changes were observed in spontaneous and stimulus-induced electric field responses. After exposing the slices to HEPES medium for at least 2 hours, perfusion was switched to 4-AP-containing HEPES medium with reduced [Cl ⁻ ] _o to 21 mM. Exposure to low- [Cl ⁻ ] _o HEPES medium induced the same sequence of events and time courses previously observed with low- [Cl ⁻ ] _o NaHCO ₃ containing medium. After complete block of spontaneous bursting, the perfusion medium normal - it returned to HEPES containing _{o ^[Cl].} The spontaneous burst resumed within 20-40 minutes. When the spontaneous burst resumed, the slices were superperfused for 3 hours in HEPES medium, which is currently free of bicarbonate.

  This data suggests that the effect of chloride cotransport antagonism on synchrony and excitability is independent of the effect on the dynamic characteristics of extracellular pH.

FIG. 4 shows a schematic model of ion cotransport under reduced [Cl ⁻ ] conditions. The left panel of FIG. 4A shows that ionic flux through the furosemide-sensitive K ⁺ , C ⁻ cotransporter maintains the chloride gradient required for IPSP generation in neurons. Under normal conditions, high concentrations of intracellular potassium (maintained by 3Na ⁺ , 2K ⁺ -ATPase pump) act as a driving force for excretion of Cl ⁻ against its concentration gradient. In glial cells, as shown in the right panel of FIG. 4A, the movement of ions through the furosemide-sensitive NKCC cotransporter reaches from the extracellular space to the intracellular space. The ion gradient required for this co-transport is partly maintained by the “transmembrane sodium cycle”: sodium ions incorporated into glial cells via NKCC co-transport maintain a low intracellular sodium concentration Thus, it is continuously discharged by a 3Na ⁺ , 2K ⁺ -ATPase pump. The velocity and direction of the ion flux through the furosemide-dependent cotransporter, neuronal ^K +, Cl ^- with respect cotransporter _{^{[K +] i x [Cl}} -] i - [K +] o x [Cl -] as _o), and with respect to NKCC cotransporter _{^{[Na +] i x [K}} +] i x [Cl -] 2 i - [Na +] O x [K +] o x [Cl -] 2 o) described as Is functionally proportional to its ionic product difference. The sign of this ion product difference indicates the direction of ion transport, and positive is from the intracellular to the extracellular space.

FIG. 4B shows a schematic phenomenological model illustrating the emergence of late spontaneous electric field events that rise as a result of prolonged low- [Cl ⁻ ] _o exposure. We denote the ionic product differences for neurons and glia as QN and QG, respectively. Under control conditions (1), the difference in ionic product for neurons is that when K ⁺ and C ⁻ are co-transported from inside the cell to the extracellular space (QN>0); The difference is that when Na ⁺ , K ⁺ and C ⁻ are co-transported from ECS to the intracellular compartment (QG <0). As [Cl ⁻ ] _o decreases (2), the ionic product difference changes such that the KCl neuronal efflux increases; the nerves so that there is a substantial efflux of KCl and NaCl from the intracellular to the extracellular space. The ion co-transport of the glue is reversed (QG> 0). These changes cause accumulation of extracellular potassium over time. Eventually, [K ⁺ ] _o reaches a level that induces depolarization of the neuronal population, resulting in a larger amount of [K ⁺ ] _o accumulation. This large accumulation of extracellular ions then functions to reverse the ion product difference so that KCl moves from the extracellular to the intracellular space (QN <0, QG <0) (3). Subsequent clearance of extracellular potassium eventually returns the transmembrane ion gradient to its initial state. By repeating this process periodically, repeated negative electric field events occur.

Example 82
Therapeutic effect of furosemide in alleviating pain symptoms in animal models of neuropathic pain The ability of furosemide to relieve pain is examined in rodents using the Chung model (eg, Walker et al. Mol. Med. Today 5: 319). -321, 1999). Sixteen adult male Long-Evans rats are used in this study. All rats are subjected to spinal cord ligation of the L5 nerve as detailed below. Eight of the 16 rats will be injected with furosemide (intravenous) and the remaining 8 will be injected intravenously with vehicle alone. The pain threshold is rapidly tested using a mechanical footpad flexion test. Compare pain threshold differences between the two groups. When furosemide relieves pain, the furosemide administration group shows a higher pain threshold than the vehicle administration group.

Chung model of neuropathy Spinal nerve ligation is performed under isoflurane anesthesia with the animal in the prone position to contact the left L4-L6 spinal nerve. Under magnification, remove about one third of the lateral protrusions. The L5 spinal nerve is identified and the adjacent L4 spinal nerve is carefully excised and then strongly ligated using 6-0 silk suture. The wound is treated with antiseptic solution, the muscle layers are ligated, and the incision is closed with a wound clip. A mechanical toe flexion threshold behavioral test is performed within 3-7 days after incision. If drowning, animals are placed in a plexiglass chamber (20 × 10.5 × 40.5 cm) and allowed to acclimate for 15 minutes. Because the chamber was placed on top of the mesh screen, mechanical stimulation can be applied to the plantar surfaces of both hind limbs. Mechanical threshold measurements for each hind limb are obtained using the up / down method with 8 von Frey monofilaments (5, 7, 13, 26, 43, 64, 106, and 202 mN). Each test begins with a von Frey force of 13 mN delivered to the right hind limb and then the left hind limb for approximately 1 second. If there is no flex response, the next higher force is delivered. If there is a response, deliver the next lower force. This procedure is carried out until there is no response at maximum force (202 mN) or until four stimuli are given after the initial response. The 50% toe flexion threshold for each toe is calculated using the following formula: [Xth] log = [vFr] log + ky, where [vFr] is the last von Frey force used, where k = 0.268 is an average interval (logarithmic unit) between von Frey monofilaments, and y is a value depending on a flexion response pattern. If the animal does not respond to maximum von Frey hair (202 mN), y = 1.00 and the 50% mechanical toe flexion response to the toes is calculated to be 340.5 mN. The mechanical footpad flexion threshold test is performed 3 times and the 50% flexion values are averaged through the 3 tests to determine the average mechanical footpad flexion threshold for each animal's right and left footpad.

Example 83
Therapeutic effects of furosemide and bumetanide in alleviating pain symptoms in severe anxiety or post-traumatic stress disorder. Therapeutic efficacy of furosemide and bumetanide in the treatment of post-traumatic stress disorder for relieving severe anxiety in contextual fear conditioning in rats Test by determining the ability of these compounds.

  Contextual fear conditioning requires a combination of aversive events, in this case moderate foot shock, and a unique environment. The intensity of fear memory is assessed using freezing, a species-specific defensive response in rats characterized by complete immobility except for breathing. Rats are placed in a unique environment and if they are immediately shocked, they do not learn fear from context. However, if allowed to investigate a unique environment prior to an immediate shock, it shows intense anxiety and fear when returned to the same environment. We can take advantage of this fact, either by learning the association between context and shock by procedurally dividing contextual fear conditioning into two phases or (emotion response circuit including amygdala) Experience the friability of shock (depending on), so that the therapeutic effects on memory on context (especially hippocampal processes) can be studied separately. Human post-traumatic stress disorder (PTSD) has been shown to be associated with emotional response circuits in the amygdala, and for this reason contextual memory conditioning is a widely acceptable model for PSTD.

  24 rats were used in the experiment. Each rat received a single 5-minute episode of a small new environmental study. After 72 hours, the rats were placed in the same environment and immediately received two moderate foot shocks (1 milliamp) separated by 53 seconds. After 24 hours, 8 rats were injected intravenously with furosemide (100 mg / kg) in vehicle (DMSO) and 8 rats were intravenously injected with bumetanide (50 mg / kg) in vehicle (DMSO). The remaining 8 animals were injected with DMSO alone. As an indicator of Pavlov's conditioned fear, each rat was placed again in the same environment for 8 minutes while freezing behavior was measured.

Four identical chambers (20 × 20 × 15 cm) were used. All situations of timing and event control were under the control of a microcomputer (MedPC, MedAssociates Inc. Vermont, USA). The measurement of freezing behavior was performed through an overhead video camera connected to a microcomputer and was automatically scored using free software, FreezeFrame ^™ (OER Inc., Reston, VA). Total freezing behavior time was analyzed in a one-way analysis of variance (ANOVA) test using drug administration as an intra-group factor.

  As shown in FIG. 5, animals treated with either bumetanide or furosemide showed significantly less freezing behavior than animals given vehicle alone, and bumetanide and furosemide are effectively used to treat posttraumatic stress disorder. Showed you get.

Example 84
Therapeutic Effects of Furosemide and Bumetanide in Relieving Anxiety The therapeutic effects of furosemide and bumetanide in relieving anxiety were examined by evaluating the effects of these substances in the Fear-Enhanced Startle (FPS) test in rats. This test is usually used to distinguish the drug effect of anxiolytics from general effects such as analgesia / muscle relaxation.

  Twenty-four rats were acclimated to the FPS apparatus for 30 minutes. After 24 hours, baseline startle amplitudes were collected. The rats were then divided into three equal groups based on the baseline startle amplitude. One rat had a significantly higher standard of startle than the other and was excluded from the experiment. Groups 1 and 2 therefore consisted of 8 rats each and Group 3 consisted of 7 rats. After baseline startle amplitude collection, 20 light / shock combinations were delivered in two periods over two consecutive days. (Eg 10 light / shock combinations each day). On the last day (day 5), groups 2 and 3 were injected (intravenously) with either furosemide (100 mg / kg) or bumetanide (70 mg / kg) in vehicle (DMSO), group 1 with vehicle alone did. Immediately after injection, startle amplitude was assessed during the startle alone test and startle plus fear (light after startle) test. Fear-enhancing startle (light + startle amplitude minus startle alone amplitude) was compared between treatment groups.

  The animals were trained and tested in 4 identical stabilizer meter devices (Med-Associates). In other words, each rat was placed in a small plexiglass cylinder. Each Stabilometer floor consisted of four 6mm diameter stainless steel bars placed 18mm apart through which shocks could be delivered. The movement of the cylinder becomes the displacement of the accelerometer, and the voltage obtained is proportional to the speed of the cage displacement. The startle amplitude is defined as the maximum accelerometer voltage that occurs during the first 0.25 seconds after delivering the startle stimulus. The accelerometer analog output was amplified, digitized to a scale of 0-4096 units, and stored on a microcomputer. Each stabilizer was ventilated and enclosed in a light and sound attenuated box. All sound level measurements were performed with a precision sound level meter. The noise of the ventilation fan mounted on the side of each wooden box produced an overall background noise level of 64 dB. The startle stimulus was a 50 ms burst of white noise (increase-decay time 5 ms) generated by a white noise generator. The visual conditioning stimulus used was light bulb illumination adjacent to a white noise source. The unconditioned stimulus was a 0.5 second 0.6 mA foot shock generated by four constant current shock absorbers placed outside the chamber. All stimulation runs and sequences were under microcomputer control.

  The FPS maneuver consisted of a 5-day study; for 1 and 2 days, baseline startle responses were collected, delivering the light / shock combination on days 3 and 4, and for fear-enhanced startle on day 5 The test was conducted.

  Matching: On the first 2 days, all rats were placed in plexiglass cylinders and after 3 minutes, 30 startle stimuli were applied with a 30 second stimulation interval. A 105 dB strength was used. The average startle amplitude during 30 startle stimuli on day 2 was used to assign rats to treatment groups in a similar manner.

  Training: The next two days, rats were placed in plexiglass cylinders. Each day, 10 CS-shock combinations were delivered 3 minutes after introduction. Shocks were delivered during the last 0.5 seconds of a CS of 3.7 seconds at an average test interval of 4 minutes (range 3-5 minutes).

  Test: Rats were placed in the same startle box where they were trained and after 3 minutes, 18 startle-stimulating stimuli were applied (all at 105 dB). These initial startle stimuli were used to re-acclimate the rats to acoustic startle stimuli. Thirty seconds after the end of these stimuli, each animal was a stimulus given half alone (startle alone test) and the other half given 3.2 seconds after the onset of the 3.7 second CS. (CS-startle stimulation), received 60 stimulations. All startle stimuli were given at an average 30 second stimulation interval that randomly varied between 20 and 40 seconds.

  Measurement: Treated groups were compared by the difference in startle amplitude between CS-startle and startle alone tests (fear synergy).

  FIG. 6 shows the baseline startle amplitude for each group of rats determined prior to the test date. FIG. 7 shows the amplitude of response in the startle alone test determined immediately after injection with DMSO alone, bumetanide or furosemide on the test day, and FIG. 8 shows the difference score on the test day (startle alone-fear enhanced startle) . As shown in the figure, statistically significantly lower difference scores were observed in rats treated with either furosemide or bumetanide than rats treated with vehicle alone, indicating that both furosemide and bumetanide have anxiety-reducing effects. Yes.

  Figures 9 and 10 show the startle alone amplitude and difference score one week after treatment with either furosemide or bumetanide, respectively. Animals treated with either furosemide or bumetanide were found to have higher differential scores than animals treated with vehicle alone. However, the error bars for vehicle-treated animals are quite large, so the data show no statistically significant difference between vehicle and bumetanide and barely show a slight difference between vehicle and furosemide.

  FIG. 12 shows either vehicle alone (n = 13) or bumetanide 10 mg (n = 13), 30 mg (n = 13), 40 mg (n = 5), 50 mg (n = 13) or 70 mg (n = 13). Difference scores for treated animals are shown. It was found that administration of 40 mg, 50 mg and 70 mg had a more remarkable anxiety reducing effect than administration of 10 mg or 30 mg.

Example 85
Therapeutic effects of bumetanide analogs in reducing anxiety The therapeutic effects of various bumetanide analogs in reducing anxiety were examined using the fear-enhanced startle (FPS) test in rats described above.

  FIG. 11 shows the following bumetanide analogs: bumetanide N, N-diethylglycolamide ester (referred to as 2MIK053); bumetanide methyl ester (referred to as 3MIK054); bumetanide N, N-dimethylglycolamide ester (as 3MIK069-11) Bumetanide morpholino diethyl ester (referred to as 3MIK066-4); bumetanide pibaxetyl ester (referred to as 3MIK069-12); bumetanide cyanomethyl ester (referred to as 3MIK047); bumetanide dibenzylamide (See 3MIK065); or differential score (startle alone-fright enhanced startle) in test days in rats treated with one of bumetanide 3- (dimethylaminopropyl) ester (see 3MIK066-5) It is. The vehicle was DMSO. As shown in FIG. 11, the differential scores obtained after administration of most bumetanide analogs are significantly lower than those observed after vehicle alone, and these analogs can be used effectively to reduce anxiety. showed that.

  In another experiment, rats were injected with either bumetanide, saline or a bumetanide analog and their urine output was measured. As shown in FIG. 13, bumetanide N, N-diethylglycolamide ester (column 2), bumetanide pibaxetyl ester (column 3) and bumetanide cyanomethyl ester (column 4) were obtained from two different raw materials. The diuretic effect was significantly lower than the bumetanide obtained (columns 1 and 13). Column 6 shows the results obtained after administration of saline.

  Although the present invention has been described with reference to specific embodiments thereof as unusual, it will be appreciated by those skilled in the art that various changes can be made and equivalents substituted without departing from the spirit and scope of the invention. In addition, many modifications may be made to a particular situation, material, material composition, method, method step, for use in the practice of the present invention. All such modifications are intended to be within the scope of the appended claims.

  All patents and publications cited herein and PCT application WO 00/37616 published June 29, 2000 are incorporated herein by reference in their entirety.

  SEQ ID NOs: 1-2 are shown in the attached sequence listing. The polynucleotide and polypeptide sequence codes used in the attached sequence listing are WIPO Standard ST. 25 (1988), Appendix2.

Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 1A, 1A1, 1B, 1B1, 1C, 1C1 and 1D show the effect of furosemide on stimuli elicited after discharge activity in hippocampal slices. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. Figures 2A-2R show the furosemide block of spontaneous soot-like burst discharge across the spectrum of the in vitro model. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 3A to 3H show the kainic acid-induced furosemide block in urethane anesthetized rats, the upper chart shows the EKG recording results and the lower chart shows the cortical EEG recording results. 4A and 4B show schematic diagrams of ion cotransport under conditions of reduced chloride concentration. 4A and 4B show schematic diagrams of ion cotransport under conditions of reduced chloride concentration. FIG. 5 shows that significantly lower freezing was observed in animals receiving either bumetanide or furosemide than in animals receiving vehicle alone in a contextual fear conditioning study in rats. FIG. 6 shows baseline startle amplification in a fear-enhanced startle test in rats. FIG. 7 shows the amplification of the rat response to the startle alone test measured immediately after administration of DMSO alone, either bumetanide or furosemide. FIG. 8 shows the difference score (startle alone-fright enhanced startle) on the test day of rats administered with DMSO, bumetanide or furosemide. FIG. 9 shows the startle-only amplification of rats one week after administration of either DMSO, bumetanide or furosemide. FIG. 10 shows the difference score one week after administration of DMSO, bumetanide or furosemide. FIG. 11 shows the following bumetanide analogs: bumetanide N, N-diethylglycolamide ester (referred to as 2MIK053); bumetanide methyl ester (referred to as 3MIK054); bumetanide N, N-dimethylglycolamide ester (3MIK069-11) Bumetanide morpholino diethylster (referred to as 3MIK066-4); bumetanide pibaxetyl ester (referred to as 3MIK069-12); bumetanide cyanomethyl ester (referred to as 3IK047); bumetanide dimendyl Figure 2 shows the differential score (startle alone-fear-enhanced startle) on the test day of rats dosed with amide (referred to as 3MIK065); and bumetanide 3- (dimethyl-aminopropyl) ester (referred to as 3MIK066-5). The vehicle was DMSO. FIG. 12 shows the differential scores for rats administered with various doses of bumetanide. FIG. 13 shows bumetanide (columns 1 and 5), bumetanide N, N-diethylglycolamide ester (column 2), bumetanide pibaxetyl ester (column 3), bumetanide cyanomethyl derived from two different raw materials. Shown are urine output in rats after administration of either ester (column 4) or saline (column 6).

Claims (20)

A method for treating an anxiety disorder in a subject comprising administering to a mammalian subject an effective amount of a composition comprising a Na ⁺ K ⁺ 2Cl cotransporter antagonist. The method of claim 1, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist reduces or blocks oversynchronous neuronal population discharge due to non-synaptic action. The method of claim 1, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist is an NKCC1 cotransporter antagonist. The method of claim 1, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist is a CNS targeting NKCC cotransporter antagonist. 5. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is selected from the group consisting of furosemide; bumetanide; ethacrynic acid; tolsemide; azosemide; muzolimine; piretanide; tripamide; and their functional analogs and derivatives. the method of. The method of claim 1, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist is selected from the group consisting of thiazide; and a thiazide-like diuretic. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is represented by the following formula I:

Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
Where
R ₁ is absent, H or O;
R ₂ is H, or when R ₁ is O, alkylaminodialkyl, alkylaminocarbonyldialkyl, alkyloxycarbonylalkyl, alkylaldehyde, alkyl ketone alkyl, alkylamide, alkylammonium group, alkylcarboxylic acid Selected from the group consisting of unsubstituted or substituted alkylheteroaryl, alkylhydroxy, biocompatible polymer, methyloxyalkyl, methyloxyalkaryl, methylthioalkylalkyl and methylthioalkaryl, and R ₁ is present If not, R ₂ is hydrogen, dialkylamino, diarylamino, dialkylaminodialkyl, dialkylcarbonylaminodialkyl, dialkyl ester alkyl, dialkyl aldehyde, dialkyl ketone Selected from the group consisting of unsubstituted or substituted alkyl, dialkylamide, dialkylcarboxylic acid and dialkylheteroaryl;
R ₃ is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy unsubstituted or substituted;
The method of claim 1, wherein R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, alkylhydroxyaminodialkyl unsubstituted or substituted. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is bumetanide aldehyde; bumetanide dibenzylamide; bumetanide diethylamide; bumetanide morpholinoethyl ester; bumetanide 3- (dimethylaminopropyl) ester; bumetanide N, N 8. The diethyl glycolamide ester; bumetanide dimethyl glycolamide ester; bumetanide pibaxetyl ester; bumetanide benzyltrimethylammonium salt; and bumetanide cetyltrimethylammonium salt. the method of. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is represented by the following formula II:

Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
Where
R ₁ is absent, H or O;
R ₂ is H, or when R ₁ is O, alkylaminodialkyl, alkylaminocarbonyldialkyl, alkyloxycarbonylalkyl, alkylaldehyde, alkyl ketone alkyl, alkylamide, alkylammonium group, alkylcarboxylic acid Selected from the group consisting of unsubstituted or substituted alkylheteroaryl, alkylhydroxy, biocompatible polymer, methyloxyalkyl, methyloxyalkaryl, methylthioalkylalkyl and methylthioalkaryl, and R ₁ is present If not, R ₂ is hydrogen, dialkylamino, diarylamino, dialkylaminodialkyl, dialkylcarbonylaminodialkyl, dialkyl ester alkyl, dialkyl aldehyde, dialkyl ketone Selected from the group consisting of unsubstituted or substituted alkyl, dialkylamide, dialkylcarboxylic acid and dialkylheteroaryl;
R ₃ is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy unsubstituted or substituted;
The method of claim 1, wherein R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, alkylhydroxyaminodialkyl unsubstituted or substituted. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is furosemide aldehyde; furosemide ethyl ester; furosemide cyanomethyl ester; furosemide benzyl ester; furosemide morpholino ethyl ester; furosemide 3- (dimethylaminopropyl) ester; furosemide N, N-diethyl glycol Furosemide dibenzylamide; Furosemide benzylmethylammonium salt; Furosemide cetyltrimethylammonium salt; Furosemide N, N-dimethylglycolamide ester; Furosemide pibaxetyl ester; and Furosemide propaxetyl ester; The method of claim 9. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is represented by the following formula III:

Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
Where
R ₁ is absent, H or O;
R ₂ is H, or when R ₁ is O, alkylaminodialkyl, alkylaminocarbonyldialkyl, alkyloxycarbonylalkyl, alkylaldehyde, alkyl ketone alkyl, alkylamide, alkylammonium group, alkylcarboxylic acid Selected from the group consisting of unsubstituted or substituted alkylheteroaryl, alkylhydroxy, biocompatible polymer, methyloxyalkyl, methyloxyalkaryl, methylthioalkylalkyl and methylthioalkaryl, and R ₁ is present If not, R ₂ is hydrogen, dialkylamino, diarylamino, dialkylaminodialkyl, dialkylcarbonylaminodialkyl, dialkyl ester alkyl, dialkyl aldehyde, dialkyl ketone Selected from the group consisting of unsubstituted or substituted alkyl, dialkylamide, dialkylcarboxylic acid and dialkylheteroaryl;
R ₃ is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy unsubstituted or substituted;
The method of claim 1, wherein R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, alkylhydroxyaminodialkyl unsubstituted or substituted. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is selected from the group consisting of piretanide aldehyde; piretanide methyl ester; piretanide cyanomethyl ester; piretanide benzyl ester; piretanide morpholinoethyl ester; Piretanide N, N-diethylglycolamide ester; Piretanide diethylamide; Piretanide dibenzylamide; Piretanide benzyltrimethylammonium salt; Piretanide cetyltrimethylammonium salt; Piretanide N, N-dimethylglycolamide ester; 12. The method of claim 11, wherein the method is selected from the group consisting of piretanide pibaxetyl ester; and piretanide propaxetyl ester. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is represented by the following formula IV:

Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
Where
R ₃ is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy unsubstituted or substituted;
R ₄ and R ₅ are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, unsubstituted or substituted alkylhydroxyaminodialkyl; and
R ₆ is an unsubstituted or substituted alkyloxycarbonylalkyl, alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, biocompatible polymer, methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl. The method of claim 1, wherein the method is selected from the group consisting of: 14. The method of claim 13, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist is selected from the group consisting of a tetrazolyl substituted azosemide; an azosemide benzyltrimethylammonium salt; and an azosemidocetyltrimethylammonium salt. The Na ⁺ K ⁺ 2Cl cotransporter antagonist is represented by the following formula V:

Or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
Where
R ₇ is alkyloxycarbonylalkyl, alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, biocompatible polymer, methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl from unsubstituted or substituted ones Selected from the group consisting of; and
^- X or a bromide, chloride, fluoride, iodide, mesylate, tosylate; or, X ^- does not exist, the process of claim 1. 16. The method of claim 15, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist is a pyridine substituted torsemide quaternary ammonium salt. Said Na ⁺ K ⁺ 2Cl cotransporter antagonist is bendroflumethiazide; benzthiazide; chlorothiazide; hydrochlorothiazide; hydroflumethiazide; methylcrothiazide; polythiazide; trichloromethiazide; 7. The method of claim 6, wherein the method is selected from the group consisting of: The method of claim 1, wherein the Na ⁺ K ⁺ 2Cl cotransporter antagonist modulates extracellular ion composition and chloride gradient in nervous system tissue.   The method of claim 1, wherein the composition is delivered orally, sublingually, nasally, transdermally, intravenously, intrathecally or by inhalation.   The method of claim 1, wherein the anxiety disorder is selected from the group consisting of panic disorder; social anxiety disorder; obsessive compulsive disorder; post-traumatic stress disorder; generalized anxiety disorder; and specific phobias.

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